Methods of improving cell-based therapy

ABSTRACT

Provided are methods for improving cell-based therapies by co-administration with an agent that increases the production and or levels of epoxygenated fatty acids, as well as kits, stents and patches for co-administering stem cells with an agent that increases the production and/or levels of epoxygenated fatty acids.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) of U.S.Provisional Application No. 62/012,422, filed on Jun. 16, 2014, which ishereby incorporated herein by reference in its entirety for allpurposes.

FIELD

Provided are methods for improving the efficacy and success ofcell-based therapies by co-administration of stem cells with an agentthat increases the production and/or level of epoxygenated fatty acids,as well as kits, stents and patches for co-administering stem cells withan agent that increases the production and or levels of EETs.

BACKGROUND

Cardiovascular disease (CVD) is the leading cause of death for both menand women in the United States. Since cardiac myocytes have limitedability to regenerate, their malfunction or significant loss due toaging or diseases can lead to lethal consequences. Recent studies haveprovided exciting evidence to support the notion that stem cells mayoffer an enormous potential for regenerative therapy. However oneintractable barrier to cell therapy in all tissues is overcoming theconfounding hurdle of scars that ensue from acute and robustinflammatory responses arising during tissue injury.

CVD is the leading cause of morbidity and mortality in the US (9-12).Therapeutic strategies using cell-based therapy to combat ischemiccardiomyopathy have not produced full restorative functions (13-15).Moreover, previous studies have demonstrated that transplanted stemcells do not engraft or survive long term due to apoptosis, increasedcollagen deposition, ischemic environment, and increased inflammationrelated factors such as free radicals and cytokines in the hostmyocardium (16-18).

Arachidonic acid is released in response to tissue injury and can bemetabolized through the cyclooxygenase (COX), lipoxygenase (LOX) andcytochrome P450 (CYP450) pathways (FIG. 1). The CYP450 epoxygenasepathway generates epoxyeicosatrienoic acids (EETs) which modulate iontransport and gene expression, producing vasorelaxation,anti-inflammatory and pro-fibrinolytic effects (19). EETs are furthermetabolized by soluble epoxide hydrolases (sEH) to form thecorresponding dihydroxyeicosatrienoic acids (DHETs) with a significantreduction in anti-hypertensive and anti-inflammatory activities (19-21).

SUMMARY

In one aspect, provided are methods of increasing, improving and/orpromoting the survival, engraftment, and/or integration of transplantedstem cells in a tissue of a subject in need thereof, comprisingco-administering to the subject the stem cells with an agent thatincreases the production and/or level of epoxygenated fatty acids.

In varying embodiments, the cardiomyopathy is hypertrophiccardiomyopathy. In varying embodiments, the cardiomyopathy ishypertensive cardiomyopathy. In varying embodiments, the cardiomyopathyis diabetic cardiomyopathy. In varying embodiments, the cardiomyopathyis due to valvular heart disease. In varying embodiments, the valvularheart disease is secondary to rheumatic fever, myxomatous degenerationof the valve, or papillary muscle dysfunction. In varying embodiments,the cardiomyopathy is due to myocardial infarction. In varyingembodiments, the cardiomyopathy is due to familial hypertrophiccardiomyopathy. In varying embodiments, the cardiomyopathy is dilatedcardiomyopathy. In varying embodiments, the dilated cardiomyopathy isalcohol-induced cardiomyopathy. In varying embodiments, the dilatedcardiomyopathy is viral-induced cardiomyopathy. In varying embodiments,the dilated cardiomyopathy is familial dilated cardiomyopathy. Invarying embodiments, the dilated cardiomyopathy is idiopathiccardiomyopathy. In varying embodiments, the dilated cardiomyopathy iscaused by administration of an anti-cancer drug or exposure to a toxicagent. In varying embodiments, the administration of said stem cells andsaid agent or agents inhibits cardiac arrhythmia. In some embodiments,the arrhythmia is atrial fibrillation or atrial flutter. In someembodiments, the arrhythmia is ventricular fibrillation. In someembodiments, the arrhythmia is ventricular tachycardia.

In varying embodiments of the methods, the agent comprises one or moreepoxygenated fatty acids. In varying embodiments, the epoxygenated fattyacids are selected from the group consisting of cis-epoxyeicosantrienoicacids (“EETs”), epoxides of linoleic acid, epoxides of eicosapentaenoicacid (“EPA”), epoxides of docosahexaenoic acid (“DHA”), epoxides of thearachidonic acid (“AA”), epoxides of cis-7,10,13,16,19-docosapentaenoicacid, and mixtures thereof. In varying embodiments, the agent increasesthe production and/or levels of cis-epoxyeicosantrienoic acids (“EETs”).In varying embodiments of the methods, the agent that increases theproduction and/or level of EETs is an inhibitor of soluble epoxidehydrolase (“sEH”). In varying embodiments, the inhibitor of sEHcomprises a primary pharmacophore selected from the group consisting ofa urea, a carbamate, and an amide. In varying embodiments, the inhibitorof sEH comprises a cyclohexyl moiety, aromatic moiety, substitutedaromatic moiety or alkyl moiety attached to the pharmacophore. Invarying embodiments, the inhibitor of sEH comprises a cyclohexyl ethermoiety attached to the pharmacophore. In varying embodiments, theinhibitor of sEH comprises a phenyl ether or piperidine moiety attachedto the pharmacophore. In varying embodiments, the inhibitor of sEHcomprises a polyether secondary pharmacophore. In varying embodiments,the inhibitor of sEH has an IC50 of less than about 100 μM. In varyingembodiments of the methods, the inhibitor of sEH is selected from thegroup consisting of:

a) 3-(4-chlorophenyl)-1-(3,4-dichlorphenyl)urea or3,4,4′-trichlorocarbanilide (TCC; compound 295);

b) 12-(3-adamantan-1-yl-ureido) dodecanoic acid (AUDA; compound 700);

c) 1-adamantanyl-3-{5-[2-(2-ethoxyethoxy)ethoxy]pentyl]}urea (AEPU;compound 950);

d) 1-(1-acetypiperidin-4-yl)-3-adamantanylurea (APAU; compound 1153);

e) trans-4-[4-(3-Adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid(tAUCB; compound 1471);

f) cis-4-[4-(3-Adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid(cAUCB; compound 1686);

g)1-(1-methylsulfonyl-piperidin-4-yl)-3-(4-trifluoromethoxy-phenyl)-urea(TUPS; compound 1709);

-   -   h)        trans-4-{4-[3-(4-Trifluoromethoxy-phenyl)-ureido]-cyclohexyloxy}-benzoic        acid (tTUCB; compound 1728);

i) 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU;compound 1770);

j) 1-(1-ethylsulfonyl-piperidin-4-yl)-3-(4-trifluoromethoxy-phenyl)-urea(TUPSE; compound 2213);

k)1-(1-(cyclopropanecarbonyl)piperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea(CPTU; compound 2214);

l)trans-N-methyl-4-[4-(3-Adamantan-1-yl-ureido)-cyclohexyloxy]-benzamide(tMAUCB; compound 2225);

m)trans-N-methyl-4-[4-((3-trifluoromethyl-4-chlorophenyl)-ureido)-cyclohexyloxy]-benzamide(tMTCUCB; compound 2226);

n)cis-N-methyl-4-{4-[3-(4-trifluoromethoxy-phenyl)-ureido]-cyclohexyloxy}-benzamide(cMTUCB; compound 2228); and

o) 1-cycloheptyl-3-(3-(1,5-diphenyl-1H-pyrazol-3-yl)propyl)urea (HDP3U;compound 2247).

In varying embodiments of the methods, the inhibitor of sEH is selectedfrom the group consisting of:

a) 3-(4-chlorophenyl)-1-(3,4-dichlorphenyl)urea or3,4,4′-trichlorocarbanilide (TCC; compound 295);

b) 12-(3-adamantan-1-yl-ureido) dodecanoic acid (AUDA; compound 700);

c) 1-adamantanyl-3-{5-[2-(2-ethoxyethoxy)ethoxy]pentyl]}urea (AEPU;compound 950);

d) 1-(1-acetypiperidin-4-yl)-3-adamantanylurea (APAU; compound 1153);

e) trans-4-[4-(3-Adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid(tAUCB; compound 1471);

f) cis-4-[4-(3-Adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid(cAUCB; compound 1686);

g)trans-4-{4-[3-(4-Trifluoromethoxy-phenyl)-ureido]-cyclohexyloxy}-benzoicacid (tTUCB; compound 1728);

h)trans-N-methyl-4-[4-(3-Adamantan-1-yl-ureido)-cyclohexyloxy]-benzamide(tMAUCB; compound 2225);

i)trans-N-methyl-4-[4-((3-trifluoromethyl-4-chlorophenyl)-ureido)-cyclohexyloxy]-benzamide(tMTCUCB; compound 2226);

j)cis-N-methyl-4-{4-[3-(4-trifluoromethoxy-phenyl)-ureido]-cyclohexyloxy}-benzamide(cMTUCB; compound 2228); and

k) 1-cycloheptyl-3-(3-(1,5-diphenyl-1H-pyrazol-3-yl)propyl)urea (HDP3U;compound 2247).

In varying embodiments of the methods, the inhibitor of sEH is selectedfrom the group consisting of:

a) trans-4-[4-(3-Adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid(tAUCB; compound 1471);

b) 4-{4-[3-(4-Trifluoromethoxy-phenyl)-ureido]-cyclohexyloxy}-benzoicacid (tTUCB; compound 1728);

c) 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU;compound 1770);

d)trans-2-(4-(4-(3-(4-trifluoromethoxy-phenyl)-ureido)-cyclohexyloxy)-benzamido)-aceticacid (compound 2283);

e)N-(methylsulfonyl)-4-(trans-4-(3-(4-trifluoromethoxy-phenyl)-ureido)-cyclohexyloxy)-benzamide(compound 2728);

f)1-(trans-4-(4-(1H-tetrazol-5-yl)-phenoxy)-cyclohexyl)-3-(4-(trifluoromethoxy)-phenyl)-urea(compound 2806);

g) 4-(trans-4-(3-(2-fluorophenyl)-ureido)-cyclohexyloxy)-benzoic acid(compound 2736);

h) 4-(4-(3-(4-(trifluoromethoxy)-phenyl)-ureido)-phenoxy)-benzoic acid(compound 2803);

i)4-(3-fluoro-4-(3-(4-(trifluoromethoxy)-phenyl)-ureido)-phenoxy)-benzoicacid (compound 2807);

j)N-hydroxy-4-(trans-4-(3-(4-(trifluoromethoxy)-phenyl)-ureido)-cyclohexyloxy)-benzamide(compound 2761);

k) (5-methyl-2-oxo-1,3-dioxol-4-yl)methyl4-((1r,4r)-4-(3-(4-(trifluoromethoxy)-phenyl)-ureido)-cyclohexyloxy)-benzoate(compound 2796);

l) 1-(4-oxocyclohexyl)-3-(4-(trifluoromethoxy)-phenyl)-urea (compound2809);

m) methyl4-(4-(3-(4-(trifluoromethoxy)-phenyl)-ureido)-cyclohexylamino)-benzoate(compound 2804);

n)1-(4-(pyrimidin-2-yloxy)-cyclohexyl)-3-(4-(trifluoromethoxy)-phenyl)-urea(compound 2810); and

o)4-(trans-4-(3-(4-(difluoromethoxy)-phenyl)-ureido)-cyclohexyloxy)-benzoicacid (compound 2805).

In varying embodiments of the methods, the inhibitor of sEH is selectedfrom the group consisting of:

a) trans-4-[4-(3-Adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid(tAUCB; compound 1471); and

b) 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU;compound 1770).

In varying embodiments of the methods, the inhibitor of sEH is selectedfrom the group consisting of:

a)trans-4-{4-[3-(4-Trifluoromethoxy-phenyl)-ureido]-cyclohexyloxy}-benzoicacid (tTUCB; compound 1728);

b) 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU;compound 1770); and

c)1-(1-methylsulfonyl-piperidin-4-yl)-3-(4-trifluoromethoxy-phenyl)-urea(TUPS; compound 1709).

In varying embodiments of the methods, the inhibitor of sEH isco-administered at a subtherapeutic dose. In varying embodiments, thesubject is a human. In varying embodiments, the stem cells are selectedfrom multipotent stem cells, pluripotent stem cells, and inducedpluripotent stem cells. In varying embodiments, the stem cells comprisemesenchymal stem cells. In varying embodiments, the stem cells comprisemyocyte stem cells. In some embodiments, the stem cells comprisecardiomyocyte stem cells. In some embodiments, the stem cells compriseadult cardiac stem cells or cardiac progenitor cells derived from humancardiac tissues. In some embodiments, the stem cells are selected fromthe group consisting of cardiomyocytes or cardiac progenitor cellsderived from multipotent stem cells, cardiomyocytes or cardiacprogenitor cells derived from pluripotent stem cells, cardiomyocytes orcardiac progenitor cells derived from induced pluripotent stem cells,adult cardiac progenitor cells and cardiac stem cells. In someembodiments, the stem cells are syngeneic to the subject. In someembodiments, the stem cells are allogeneic to the subject. In someembodiments, the stem cells are xenogeneic to the subject. In someembodiments, at least about 1 million (1×10⁶) to about 1 billion (1×10⁹)stem cells are administered. In some embodiments, the stem cells areadministered intravenously, intra-arterially or intralesionally. In someembodiments, the stem cells and the agent that increases the productionand/or level of epoxygenated fatty acids are administered by the sameroute of administration. In some embodiments, the stem cells and theagent that increases the production and/or level of epoxygenated fattyacids are administered by different routes of administration. In someembodiments, the stem cells and the inhibitor of sEH are concurrentlyco-administered. In some embodiments, the stem cells and the inhibitorof sEH are sequentially co-administered. In some embodiments, the tissueis cardiac tissue.

In another aspect, provided are kits, stents and patches. In someembodiments, the kits, stents and patches comprise a population of stemcells and one or more agents that increase the production and/or levelof epoxygenated fatty acids. In varying embodiments, of the stents, thestent is a coronary artery stent. In varying embodiments of the patches,the patch is a cardiac patch.

In varying embodiments of the kits, stents and patches, the agentcomprises one or more epoxygenated fatty acids. In varying embodiments,the epoxygenated fatty acids are selected from the group consisting ofcis-epoxyeicosantrienoic acids (“EETs”), epoxides of linoleic acid,epoxides of eicosapentaenoic acid (“EPA”), epoxides of docosahexaenoicacid (“DHA”), epoxides of the arachidonic acid (“AA”), epoxides ofcis-7,10,13,16,19-docosapentaenoic acid, and mixtures thereof. Invarying embodiments, the agent increases the production and/or levels ofcis-epoxyeicosantrienoic acids (“EETs”). In varying embodiments of thekits, stents and patches, the agent that increases the production and/orlevel of EETs is an inhibitor of soluble epoxide hydrolase (“sEH”). Invarying embodiments, the inhibitor of sEH comprises a primarypharmacophore selected from the group consisting of a urea, a carbamate,and an amide. In varying embodiments, the inhibitor of sEH comprises acyclohexyl moiety, aromatic moiety, substituted aromatic moiety or alkylmoiety attached to the pharmacophore. In varying embodiments, theinhibitor of sEH comprises a cyclohexyl ether moiety attached to thepharmacophore. In varying embodiments, the inhibitor of sEH comprises aphenyl ether or piperidine moiety attached to the pharmacophore. Invarying embodiments, the inhibitor of sEH comprises a polyethersecondary pharmacophore. In varying embodiments, the inhibitor of sEHhas an IC50 of less than about 100 μM. In varying embodiments of thekits, stents and patches, the inhibitor of sEH is selected from thegroup consisting of:

a) 3-(4-chlorophenyl)-1-(3,4-dichlorphenyl)urea or3,4,4′-trichlorocarbanilide (TCC; compound 295);

b) 12-(3-adamantan-1-yl-ureido) dodecanoic acid (AUDA; compound 700);

c) 1-adamantanyl-3-{5-[2-(2-ethoxyethoxy)ethoxy]pentyl]}urea (AEPU;compound 950);

d) 1-(1-acetypiperidin-4-yl)-3-adamantanylurea (APAU; compound 1153);

e) trans-4-[4-(3-Adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid(tAUCB; compound 1471);

f) cis-4-[4-(3-Adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid(cAUCB; compound 1686);

g)1-(1-methylsulfonyl-piperidin-4-yl)-3-(4-trifluoromethoxy-phenyl)-urea(TUPS; compound 1709);

h)trans-4-{4-[3-(4-Trifluoromethoxy-phenyl)-ureido]-cyclohexyloxy}-benzoicacid (tTUCB; compound 1728);

i) 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU;compound 1770);

j) 1-(1-ethylsulfonyl-piperidin-4-yl)-3-(4-trifluoromethoxy-phenyl)-urea(TUPSE; compound 2213)

k)1-(1-(cyclopropanecarbonyl)piperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea(CPTU; compound 2214);

l)trans-N-methyl-4-[4-(3-Adamantan-1-yl-ureido)-cyclohexyloxy]-benzamide(tMAUCB; compound 2225);

m)trans-N-methyl-4-[4-((3-trifluoromethyl-4-chlorophenyl)-ureido)-cyclohexyloxy]-benzamide(tMTCUCB; compound 2226);

n)cis-N-methyl-4-{4-[3-(4-trifluoromethoxy-phenyl)-ureido]-cyclohexyloxy}-benzamide(cMTUCB; compound 2228); and

o) 1-cycloheptyl-3-(3-(1,5-diphenyl-1H-pyrazol-3-yl)propyl)urea (HDP3U;compound 2247).

In varying embodiments of the kits, stents and patches, the inhibitor ofsEH is selected from the group consisting of:

a) 3-(4-chlorophenyl)-1-(3,4-dichlorphenyl)urea or3,4,4′-trichlorocarbanilide (TCC; compound 295);

b) 12-(3-adamantan-1-yl-ureido) dodecanoic acid (AUDA; compound 700);

c) 1-adamantanyl-3-{5-[2-(2-ethoxyethoxy)ethoxy]pentyl]}urea (AEPU;compound 950);

d) 1-(1-acetypiperidin-4-yl)-3-adamantanylurea (APAU; compound 1153);

e) trans-4-[4-(3-Adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid(tAUCB; compound 1471);

f) cis-4-[4-(3-Adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid(cAUCB; compound 1686);

g)trans-4-{4-[3-(4-Trifluoromethoxy-phenyl)-ureido]-cyclohexyloxy}-benzoicacid (tTUCB; compound 1728);

h)trans-N-methyl-4-[4-(3-Adamantan-1-yl-ureido)-cyclohexyloxy]-benzamide(tMAUCB; compound 2225);

i)trans-N-methyl-4-[4-((3-trifluoromethyl-4-chlorophenyl)-ureido)-cyclohexyloxy]-benzamide(tMTCUCB; compound 2226);

j)cis-N-methyl-4-{4-[3-(4-trifluoromethoxy-phenyl)-ureido]-cyclohexyloxy}-benzamide(cMTUCB; compound 2228); and

-   -   k) 1-cycloheptyl-3-(3-(1,5-diphenyl-1H-pyrazol-3-yl)propyl)urea        (HDP3U; compound 2247).

In varying embodiments of the kits, stents and patches, the inhibitor ofsEH is selected from the group consisting of:

a) trans-4-[4-(3-Adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid(tAUCB; compound 1471);

b) 4-{4-[3-(4-Trifluoromethoxy-phenyl)-ureido]-cyclohexyloxy}-benzoicacid (tTUCB; compound 1728);

c) 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU;compound 1770);

d)trans-2-(4-(4-(3-(4-trifluoromethoxy-phenyl)-ureido)-cyclohexyloxy)-benzamido)-aceticacid (compound 2283);

e)N-(methylsulfonyl)-4-(trans-4-(3-(4-trifluoromethoxy-phenyl)-ureido)-cyclohexyloxy)-benzamide(compound 2728);

f)1-(trans-4-(4-(1H-tetrazol-5-yl)-phenoxy)-cyclohexyl)-3-(4-(trifluoromethoxy)-phenyl)-urea(compound 2806);

g) 4-(trans-4-(3-(2-fluorophenyl)-ureido)-cyclohexyloxy)-benzoic acid(compound 2736);

h) 4-(4-(3-(4-(trifluoromethoxy)-phenyl)-ureido)-phenoxy)-benzoic acid(compound 2803);

i)4-(3-fluoro-4-(3-(4-(trifluoromethoxy)-phenyl)-ureido)-phenoxy)-benzoicacid (compound 2807);

j)N-hydroxy-4-(trans-4-(3-(4-(trifluoromethoxy)-phenyl)-ureido)-cyclohexyloxy)-benzamide(compound 2761);

k) (5-methyl-2-oxo-1,3-dioxol-4-yl)methyl4-((1r,4r)-4-(3-(4-(trifluoromethoxy)-phenyl)-ureido)-cyclohexyloxy)-benzoate(compound 2796);

l) 1-(4-oxocyclohexyl)-3-(4-(trifluoromethoxy)-phenyl)-urea (compound2809);

m) methyl4-(4-(3-(4-(trifluoromethoxy)-phenyl)-ureido)-cyclohexylamino)-benzoate(compound 2804);

n)1-(4-(pyrimidin-2-yloxy)-cyclohexyl)-3-(4-(trifluoromethoxy)-phenyl)-urea(compound 2810); and

o)4-(trans-4-(3-(4-(difluoromethoxy)-phenyl)-ureido)-cyclohexyloxy)-benzoicacid (compound 2805).

In varying embodiments of the kits, stents and patches, the inhibitor ofsEH is selected from the group consisting of:

a)trans-4-{4-[3-(4-Trifluoromethoxy-phenyl)-ureido]-cyclohexyloxy}-benzoicacid (tTUCB; compound 1728);

b) 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU;compound 1770); and

c)1-(1-methylsulfonyl-piperidin-4-yl)-3-(4-trifluoromethoxy-phenyl)-urea(TUPS; compound 1709).

In varying embodiments of the kits, stents and patches, the inhibitor ofsEH is selected from the group consisting of:

a) trans-4-[4-(3-Adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid(tAUCB; compound 1471); and

b) 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU;compound 1770).

In varying embodiments of the kits, stents and patches, the stem cellsare selected from multipotent stem cells, pluripotent stem cells, andinduced pluripotent stem cells. In varying embodiments, the stem cellscomprise mesenchymal stem cells. In varying embodiments, the stem cellscomprise myocyte stem cells. In varying embodiments, the stem cellscomprise cardiomyocyte stem cells. In varying embodiments, the stemcells are selected from the group consisting of cardiomyocytes orcardiac progenitor cells derived from multipotent stem cells,cardiomyocytes or cardiac progenitor cells derived from pluripotent stemcells, cardiomyocytes or cardiac progenitor cells derived from inducedpluripotent stem cells, adult cardiac progenitor cells and cardiac stemcells. In varying embodiments, the population of stem cells comprises atleast about 1 million (1×10⁶) to about 1 billion (1×10⁹) stem cells.

DEFINITIONS

Units, prefixes, and symbols are denoted in their System Internationalde Unites (SI) accepted form. Numeric ranges are inclusive of thenumbers defining the range. Unless otherwise indicated, nucleic acidsare written left to right in 5′ to 3′ orientation; amino acid sequencesare written left to right in amino to carboxy orientation. The headingsprovided herein are not limitations of the various aspects orembodiments, which can be had by reference to the specification as awhole. Accordingly, the terms defined immediately below are more fullydefined by reference to the specification in its entirety. Terms notdefined herein have their ordinary meaning as understood by a person ofskill in the art.

“cis-Epoxyeicosatrienoic acids” (“EETs”) are biomediators synthesized bycytochrome P450 epoxygenases. As discussed further in a separate sectionbelow, while the use of unmodified EETs is the most preferred,derivatives of EETs, such as amides and esters (both natural andsynthetic), EETs analogs, and EETs optical isomers can all be used inthe methods, both in pure form and as mixtures of these forms. Forconvenience of reference, the term “EETs” as used herein refers to allof these forms unless otherwise required by context.

“Epoxide hydrolases” (“EH;” EC 3.3.2.3) are enzymes in the alpha betahydrolase fold family that add water to 3-membered cyclic ethers termedepoxides.

“Soluble epoxide hydrolase” (“sEH”) is an epoxide hydrolase which inendothelial and smooth muscle cells converts EETs to dihydroxyderivatives called dihydroxyeicosatrienoic acids (“DHETs”). The cloningand sequence of the murine sEH is set forth in Grant et al., J. Biol.Chem. 268(23):17628-17633 (1993). The cloning, sequence, and accessionnumbers of the human sEH sequence are set forth in Beetham et al., Arch.Biochem. Biophys. 305(1):197-201 (1993). The amino acid sequence ofhuman sEH is SEQ ID NO.:1, while the nucleic acid sequence encoding thehuman sEH is SEQ ID NO.:2. (The sequence set forth as SEQ ID NO.:2 isthe coding portion of the sequence set forth in the Beetham et al. 1993paper and in the NCBI Entrez Nucleotide Browser at accession numberL05779, which include the 5′ untranslated region and the 3′ untranslatedregion.) The evolution and nomenclature of the gene is discussed inBeetham et al., DNA Cell Biol. 14(1):61-71 (1995). Soluble epoxidehydrolase represents a single highly conserved gene product with over90% homology between rodent and human (Arand et al., FEBS Lett.,338:251-256 (1994)). Unless otherwise specified, as used herein, theterms “soluble epoxide hydrolase” and “sEH” refer to human sEH.

Unless otherwise specified, as used herein, the term “sEH inhibitor”(also abbreviated as “sEHI”) refers to an inhibitor of human sEH.Preferably, the inhibitor does not also inhibit the activity ofmicrosomal epoxide hydrolase by more than 25% at concentrations at whichthe inhibitor inhibits sEH by at least 50%, and more preferably does notinhibit mEH by more than 10% at that concentration. For convenience ofreference, unless otherwise required by context, the term “sEHinhibitor” as used herein encompasses prodrugs which are metabolized toactive inhibitors of sEH. Further for convenience of reference, andexcept as otherwise required by context, reference herein to a compoundas an inhibitor of sEH includes reference to derivatives of thatcompound (such as an ester of that compound) that retain activity as ansEH inhibitor.

“Valvular heart disease” refers to a disorder of any one of the fourvalves of the heart. More particularly in the context of the presentinvention, it refers to conditions in which the disorder increasespressure in a chamber or chambers of the heart. For example, mitralvalve insufficiency permits some blood to flow back from the leftventricle into the left atrium rather than into the aorta, increasingthe pressure in the atrium.

“Fibrillation,” as defined on the website of the American College ofCardiology, is an abnormal, uncontrolled rapid contraction of the fibersin the heart. It further states: “When the process involves the twoupper chambers of the heart (the atria), the condition is called ‘atrialfibrillation.’ When it involves the lower, ventricular chambers, thecondition is called ‘ventricular fibrillation.’”

An “arrhythmia” is a disorder of the regular rhythmic beating of theheart. As used herein, the term refers to atrial or ventricularfibrillation.

“Arrhythmogenic right ventricular cardiomyopathy” or “ARVC” is arecently recognized form of cardiomyopathy in which electricaldisturbances affect the functioning of the right ventricle more than theleft ventricle. According to the website of the CardiomyopathyAssociation, it is defined as a heart muscle disease characterized bythe replacement of heart muscle by fibrous scar and fatty tissue, andhas acquired several names, all of which denote the same condition. Inaddition to ARVC, the most common term, it has also been calledArrhythmogenic Right Ventricular Dysplasia/Cardiomyopathy (ARVD/C) andArrhythmogenic Right Ventricular Dysplasia (ARVD). It is thought toaffect between 1:3,000 and 1:10,000 people.

By “physiological conditions” is meant an extracellular milieu havingconditions (e.g., temperature, pH, and osmolarity) which allows for thesustenance or growth of a cell of interest.

“Micro-RNA” (“miRNA”) refers to small, noncoding RNAs of 18-25 nt inlength that negatively regulate their complementary mRNAs at theposttranscriptional level in many eukaryotic organisms. See, e.g.,Kurihara and Watanabe, Proc Natl Acad Sci USA 101(34):12753-12758(2004). Micro-RNA's were first discovered in the roundworm C. elegans inthe early 1990s and are now known in many species, including humans. Asused herein, it refers to exogenously administered miRNA unlessspecifically noted or otherwise required by context.

With respect to cardiac arrhythmias, “inhibiting” means that therecurrence of such arrhythmias are reduced or eliminated, or that theduration of such arrhythmias is reduced, or both. With respect tocardiac hypertrophy or dilated cardiomyopathy, “inhibiting” means (i)the prevention of the development of the condition in a subject at riskthereof or (ii) in the case of a subject with cardiac hypertrophy, thereversal of hypertrophy.

Cytochrome P450 (“CYP450”) metabolism produces cis-epoxydocosapentaenoicacids (“EpDPEs”) and cis-epoxyeicosatetraenoic acids (“EpETEs”) fromdocosahexaenoic acid (“DHA”) and eicosapentaenoic acid (“EPA”),respectively. These epoxides are known endothelium-derivedhyperpolarizing factors (“EDHFs”). These EDHFs, and others yetunidentified, are mediators released from vascular endothelial cells inresponse to acetylcholine and bradykinin, and are distinct from the NOS-(nitric oxide) and COX-derived (prostacyclin) vasodilators. Overallcytochrome P450 (CYP450) metabolism of polyunsaturated fatty acidsproduces epoxides, such as EETs, which are prime candidates for theactive mediator(s). 14(15)-EpETE, for example, is derived viaepoxidation of the 14,15-double bond of EPA and is the ω-3 homolog of14(15)-EpETrE (“14(15)EET”) derived via epoxidation of the 14,15-doublebond of arachidonic acid.

“IC₅₀” refers to the concentration of an agent required to inhibitenzyme activity by 50%.

By “physiological conditions” is meant an extracellular milieu havingconditions (e.g., temperature, pH, and osmolarity) which allows for thesustenance or growth of a cell of interest.

“Micro-RNA” (“miRNA”) refers to small, noncoding RNAs of 18-25 nt inlength that negatively regulate their complementary mRNAs at theposttranscriptional level in many eukaryotic organisms. See, e.g.,Kurihara and Watanabe, Proc Natl Acad Sci USA 101(34):12753-12758(2004). Micro-RNA's were first discovered in the roundworm C. elegans inthe early 1990s and are now known in many species, including humans. Asused herein, it refers to exogenously administered miRNA unlessspecifically noted or otherwise required by context.

The term “therapeutically effective amount” refers to that amount of thecompound being administered sufficient to prevent or decrease thedevelopment of one or more of the symptoms of the disease, condition ordisorder being treated (e.g., fibrosis and/or inflammation).

The terms “prophylactically effective amount” and “amount that iseffective to prevent” refer to that amount of drug that will prevent orreduce the risk of occurrence of the biological or medical event that issought to be prevented. In many instances, the prophylacticallyeffective amount is the same as the therapeutically effective amount.

“Subtherapeutic dose” refers to a dose of a pharmacologically activeagent(s), either as an administered dose of pharmacologically activeagent, or actual level of pharmacologically active agent in a subjectthat functionally is insufficient to elicit the intended pharmacologicaleffect in itself (e.g., to obtain analgesic, anti-inflammatory, and/oranti-fibrotic effects), or that quantitatively is less than theestablished therapeutic dose for that particular pharmacological agent(e.g., as published in a reference consulted by a person of skill, forexample, doses for a pharmacological agent published in the Physicians'Desk Reference, 65th Ed., 2011, Thomson Healthcare or Brunton, et al.,Goodman & Gilman's The Pharmacological Basis of Therapeutics, 12thedition, 2010, McGraw-Hill Professional). A “subtherapeutic dose” can bedefined in relative terms (i.e., as a percentage amount (less than 100%)of the amount of pharmacologically active agent conventionallyadministered). For example, a subtherapeutic dose amount can be about 1%to about 75% of the amount of pharmacologically active agentconventionally administered. In some embodiments, a subtherapeutic dosecan be about 75%, 50%, 30%, 25%, 20%, 10% or less, than the amount ofpharmacologically active agent conventionally administered.

The terms “controlled release,” “sustained release,” “extended release,”and “timed release” are intended to refer interchangeably to anydrug-containing formulation in which release of the drug is notimmediate, i.e., with a “controlled release” formulation, oraladministration does not result in immediate release of the drug into anabsorption pool. The terms are used interchangeably with “nonimmediaterelease” as defined in Remington: The Science and Practice of Pharmacy,University of the Sciences in Philadelphia, Eds., 21^(st) Ed.,Lippencott Williams & Wilkins (2005).

The terms “sustained release” and “extended release” are used in theirconventional sense to refer to a drug formulation that provides forgradual release of a drug over an extended period of time, for example,12 hours or more, and that preferably, although not necessarily, resultsin substantially steady-state blood levels of a drug over an extendedtime period.

As used herein, the term “delayed release” refers to a pharmaceuticalpreparation that passes through the stomach intact and dissolves in thesmall intestine.

As used herein, “synergy” or “synergistic” interchangeably refer to thecombined effects of two active agents that are greater than theiradditive effects. Synergy can also be achieved by producing anefficacious effect with combined inefficacious doses of two activeagents. The measure of synergy is independent of statisticalsignificance.

The terms “systemic administration” and “systemically administered”refer to a method of administering agent (e.g., stem cells, an agentthat increases epoxygenated fatty acids (e.g., an inhibitor of sEH, anEET, an epoxygenated fatty acid, and mixtures thereof), optionally withan anti-inflammatory agent and/or an analgesic agent) to a mammal sothat the agent/cells is delivered to sites in the body, including thetargeted site of pharmaceutical action, via the circulatory system.Systemic administration includes, but is not limited to, oral,intranasal, rectal and parenteral (i.e., other than through thealimentary tract, such as intramuscular, intravenous, intra-arterial,transdermal and subcutaneous) administration.

The term “co-administration” refers to the presence of both activeagents/cells in the blood or body at the same time. Active agents thatare co-administered can be delivered concurrently (i.e., at the sametime) or sequentially.

The phrase “cause to be administered” refers to the actions taken by amedical professional (e.g., a physician), or a person controllingmedical care of a subject, that control and/or permit the administrationof the agent(s)/compound(s)/cell(s) at issue to the subject. Causing tobe administered can involve diagnosis and/or determination of anappropriate therapeutic or prophylactic regimen, and/or prescribingparticular agent(s)/compounds/cell(s) for a subject. Such prescribingcan include, for example, drafting a prescription form, annotating amedical record, and the like.

The term “mesenchymal stem cells” refers to stem cells defined by theircapacity to differentiate into bone, cartilage, and adipose tissue. Withrespect to cell surface markers, MSCs generally express CD44, CD90 andCD105, and do not express CD4, CD34, CD45, CD80, CD86 or MHC-II.

The terms “patient,” “subject” or “individual” interchangeably refers toa non-human mammal, including primates (e.g., macaque, pan troglodyte,pongo), a domesticated mammal (e.g., felines, canines), an agriculturalmammal (e.g., bovine, ovine, porcine, equine) and a laboratory mammal orrodent (e.g., rattus, murine, lagomorpha, hamster).

The term “mitigating” refers to reduction or elimination of one or moresymptoms of that pathology or disease, and/or a reduction in the rate ordelay of onset or severity of one or more symptoms of that pathology ordisease, and/or the prevention of that pathology or disease.

The terms “inhibiting,” “reducing,” “decreasing” refers to inhibitingthe fibrosis and/or inflammation in a non-human mammalian subject by ameasurable amount using any method known in the art. For example,inflammation is inhibited, reduced or decreased if an indicator ofinflammation, e.g., swelling, blood levels of prostaglandin PGE2, is atleast about 10%, 20%, 30%, 50%, 80%, or 100% reduced, e.g., incomparison to the same inflammatory indicator prior to administration ofan agent that increases epoxygenated fatty acids (e.g., an inhibitor ofsEH, an EET, an epoxygenated fatty acid, and mixtures thereof). In someembodiments, the fibrosis and/or inflammation is inhibited, reduced ordecreased by at least about 1-fold, 2-fold, 3-fold, 4-fold, or more incomparison to the fibrosis and/or inflammation prior to administrationof the agent that increases epoxygenated fatty acids (e.g., an inhibitorof sEH, an EET, an epoxygenated fatty acid, and mixtures thereof).Indicators of fibrosis and/or inflammation can also be qualitative.

As used herein, the phrase “consisting essentially of” refers to thegenera or species of active pharmaceutical agents included in a methodor composition, as well as any excipients inactive for the intendedpurpose of the methods or compositions. In some embodiments, the phrase“consisting essentially of” expressly excludes the inclusion of one ormore additional active agents other than the listed active agents, e.g.,an agent that increases epoxygenated fatty acids (e.g., an inhibitor ofsEH, an EET, an epoxygenated fatty acid, and mixtures thereof) and/or ananti-inflammatory agent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates arachadonic acid metabolism pathway.

FIGS. 2A-E illustrate beneficial effects of TPPU in ventricularremodeling post MI. a, Structure of the sEHI (TPPU). b, Experimentalprotocol. c, Examples of whole hearts from MI & sham mice with & withoutTPPU. d, The amount of collagen deposition in cardiac sections (Siriusred). e, Confocal images of wheat germ agglutinin showing a significantdecrease in collagen deposition in MI mice with TPPU vs. MI alone (scalebar, 50 mm). n=3 per group.

FIGS. 3A-D illustrate flow cytometric analysis of Thy1.2+ cells fromcardiac tissue. A) The selection of nucleated cells (Nucl cells) basedon the incorporation of 7AAD, the separation of myocytes (MCs) fromnon-muscle cells (NMC) and the separation of Thy1.2+ cells. B) Thy1.2+cells from the four groups of animals. C, D) Percentages of Thy1.2+cells and Ki67 positivity in Thy1.2+ cells respectively *P<0.05. n=3 pergroup. Error bars=standard error.

FIGS. 4A-B illustrate plasma levels of selected oxylipin, chemokines andcytokines A, oxylipin profiling from sham (white bars), MI (black bars)and MI treated with sEHI (t-AUCB) (gray bars) at 3 weeks of follow up.B, serum concentrations of IL-12p70, TNF-α, IFN-γ, MCP-1, and IL-6 ofsham (white bars), MI (black bars) and MI treated with sEHI (t-AUCB)(gray bars) at 3 weeks of follow up (*p<0.05).

FIG. 5 illustrate FACS of GFP+hiPSCs and confocal images of culturedGFP+hiPSCs. Scale bar=50 μM.

FIGS. 6A-C illustrate in vivo Bioluminescence (BLI) Imaging: A.

Longitudinal imaging of transplanted hiPSC-CMs (Cells) in NSG mice withsEHI treatment (bottom panel) and without sEHI treatment (top panel). B.In vitro BLI imaging of varying numbers of GFP+hiPSCs. C. Quantificationof BLI signals showing a significant increase in BLI in sEHI treatedmouse (Blue) vs non-treated mouse (red). * P<0.05.

FIGS. 7A-C illustrate transplantation of hiPSCs in NSG mice: AEchocardiogram tracings at 1 and 4 weeks post-operation in the sixgroups (Sham±sEHI, MI±sEHI, &MI+hiPSC-CM±sEHI) B. Quantification of FSin the six groups. Numbers represents FS %. Cells=hiPSC-CMs C.Ultrasound-guided injection of hiPSC-CMs in the left ventricle of themouse heart, *P<0.05.

FIG. 8 illustrates assessment of diastolic function and in vivohemodynamic analyses: Examples of pulsed-wave mitral inflow analyses(Left Panel) and pulsed-wave tissue Doppler imaging (TDI, Middle Panel)in the mouse model. Pulsed-wave TDI was performed at the lateral mitralannulus using apical 4 chamber view. Examples of PV loops (Right Panel)in a sham animal.

FIG. 9 illustrates in vivo electrophysiologic studies in sham and MImodel. Examples of inducible atrial and ventricular arrhythmias in theMI model using program stimulation (Middle and Right Panels).

FIGS. 10A-B. A) Cardiac sections stained with Trichrome demonstratingcollagen deposition. B) Immunofluorescence images of cardiac sectionsshowing the presence of GFP+ cells. Scale bar=200 μM. Cells=hiPSC-CMs.

FIGS. 11A-E illustrate second harmonic generation (SHG) signal fromhiPSC-CM. a. shows the absence of SHG signal (top right) in hiPSC (redcells labeled with Oct3/4). b, Two photon fluorescence from α-actininstain (red, left) and the SHG from myosin bundles (green, right) fromhiPSC-CM. c, A schematic representation of the SHG signal. d, A row ofsarcomeres from cardiomyocytes: SHG signal from the myosin rods (green)and RyR2 (red). e, graphs of the two photon fluorescence and SHGsignals.

FIGS. 12A-B illustrate flow cytometric analysis of Annexin V innon-myocyte (NMC) and CM from Sham±sEHI, injury±sEHI andInjury+Cells±sEHI mice (Cells=hiPSC-CM) (B) Summary data from A. *p<0.05. Error bars=SE.

FIGS. 13A-B illustrate flow cytometric analysis of Annexin V intransplanted GFP+hiPSC-CMs from MI+Cells, and MI+Cells+sEHI mice(Cells=hiPSC-CM) (B) Summary data from A. * p<0.05, Error bars=SE.

FIGS. 14A-B illustrate flow cytometric analysis of Annexin V innon-myocyte (NMC) & CM Sham±sEHI, injury±sEHI & Injury+Cells±sEHI mice(Cells=hiPSC-CM) (B) Summary data from A. * p<0.05. Error bars=SE.

FIGS. 15A-B illustrate flow cytometric analysis of ROS activity intransplanted GFP+hiPSC-CMs from MI+Cells, and MI+Cells+sEHI mice(Cells=hiPSC-CM) (B) Summary data * p<0.05, Error bars=standard error,FL=fluorescence.

FIGS. 16A-D. A) Prevention of translocation of NF-κB after treatmentwith sEHI in cultured hiPSC-CMs stimulated with TNF-α. B). Western blotanalysis of total IκB, phosphorylated IκB, nuclear NF-κB (nNF-κB) andGAPDH levels. C) & D) Quantification of Nuclear NF-κB and pIκBrespectively normalized to GAPDH. Scale bar=200 μM. *p<0.05 n=3/group.

DETAILED DESCRIPTION 1. Introduction

Provided are methods based, in part, on the discovery that theinhibition of soluble epoxide hydrolase (“sEH”), by enhancing thebiological activity of EETs with anti-inflammatory actions (22),facilitates the survival, engraftment, integration, and function oftransplanted stem cells to repair injured cardiac tissue.

The data provided herein employed a clinically relevant model ofmyocardial infarction (MI) in mice. Human induced pluripotent stemcell-derived cardiomyocytes (hiPSC-CMs) were used as the stem cellmodel. However, other types of stem cells can be used as well.Suppression of inflammation and resolution of pre-existing scar tissuesusing co-administration of sEHIs with stem cells provides an adjuvant tocell therapy independent of cells and tissue types.

Since the CYP450 pathway is evolutionarily preserved, the methods may beexploited in other inflammatory-related diseases, serving as a paradigmshift and a rehearsal for future cell-based therapy. Thus, theinnovations stem from the use of specific, novel, and potent inhibitorsof inflammation to reduce and remodel scars and to promote stem cellsurvival, engraftment, and integration. The methods further challengeconventional approaches in the treatment of MI. The data provided hereindemonstrate that these tantalizing concepts are feasible.

2. Conditions Subject to Treatment

The methods can facilitate, increase, improve and/or promote thesurvival, engraftment, and/or integration of transplanted stem cells ina tissue of an individual in need thereof, for any condition subject totreatment by administration of stem cells. For example,co-administration of stem cells with an agent that increases theproduction and/or level of cis-epoxyeicosantrienoic acids (“EETs”) canfacilitate, increase, improve and/or promote the survival, engraftment,and/or integration of transplanted stem cells to cardiac tissue, braintissue, neurological tissue (e.g., central or peripheral), musculartissue, bone tissue, renal tissue, skin, liver, pancreas, lung, innerear, spinal cord, gingiva, or any other tissue that can be healed orrepaired using stem cells.

In varying embodiments, the subject has cardiomyopathy or cardiacarrhythmia. For example, the subject may have hypertrophiccardiomyopathy, e.g., due to valvular heart disease, familialhypertrophic cardiomyopathy, dilated cardiomyopathy, myocardialinfarction, or secondary to administration of an anti-cancer drug orexposure to a toxic agent. Valvular heart disease can arise from anyetiology, including, e.g., secondary to rheumatic fever, myxomatousdegeneration of the valve, or papillary muscle dysfunction. In varyingembodiments, the subject has cardiac arrhythmia, e.g., due to atrialfibrillation, ventricular fibrillation, or ventricular tachycardia.

a. Cardiac Hypertrophy

Cardiomyocytes are terminally differentiated cells. In response tovarious extracellular stimuli, cardiomyocytes grow in a hypertrophicmanner, an event that is characterized by enlargement of individual cellsize, an increase in the content of contractile proteins such as myosinheavy chain, and expression of embryonic genes such as atrialnatriuretic factor (ANF). (Chien et al., Faseb J.; 5:3037-46 (1991);Chien, Cardiologia.; 37:95-103 (1992); Chien, J Clin Invest.;105:1339-42 (2000)) The collective result is cardiac hypertrophy, whichis an adaptive and compensatory response in nature. The initial orcompensated stage of hypertrophy normalizes wall stress per unit ofmyocardium and is thus a basic mechanism for maintaining normal chamberfunction. (Grossman et al, J Clin Invest.; 56:56-64 (1975)) However,this process is a double-edged sword: sustained cardiac hypertrophy willeventually lead to overt heart failure.

In most instances, heart failure is the final consequence of manyunderlying disease etiologies such as long-standing hypertension,coronary heart disease, valvular insufficiency, arrhythmia, viralmyocarditis, and mutations in sarcomere-encoding genes. A compensatoryenlargement of the myocardium, or hypertrophy, typically accompaniesmany of these predisposing insults and is a leading predictor for thedevelopment of more serious and life-threatening disease. Decompensatedhypertrophy occurs if increased cardiac mass fails to normalize wallstress and the contractile function is not sufficient to maintain normalpump function. This is associated with clinical and pathologicalfeatures of congestion.

Cardiac hypertrophy is characterized by an increase in heart-to-bodyweight ratio and an increase in the size of the individual cardiacmyocytes, enhanced protein synthesis, and heightened organization of thesarcomere. Classically, two different hypertrophic phenotypes can bedistinguished: (1) concentric hypertrophy due to pressure overload,which is characterized by parallel addition of sarcomeres and lateralgrowth of individual cardiomyocytes, and (2) eccentric hypertrophy dueto volume overload or prior infarction, characterized by addition ofsarcomeres in series and longitudinal cell growth. (Dorn et al., CircRes.; 92:1171-5 (2003)). At the molecular level, these changes incellular phenotype are accompanied by reinduction of the so-called fetalgene program, because patterns of gene expression mimic those seenduring embryonic development. (Chien et al., Faseb J; 5:3037-46 (1991);Chien K R, Cardiologia.; 37:95-103 (1992)).

Hypertrophic transformation of the heart can be divided into threestages: (1) developing hypertrophy, in which load exceeds output, (2)compensatory hypertrophy, in which the workload/mass ratio is normalizedand resting cardiac output is maintained, and (3) overt heart failure,with ventricular dilation and progressive declines in cardiac outputdespite continuous activation of the hypertrophic program. (Meerson F Z,Cor Vasa.; 3:161-77 (1961)). The late-phase “remodeling” process thatleads to failure is associated with functional perturbations of cellularCa²⁺ homeostasis (Bers D M, Nature.; 415:198-205 (2002); Bers D M, CircRes.; 90:14-7 (2002)) and ionic currents, (Ahmmed et al., Circ Res.;86(5):558-70 (2000); Kaab et al., Circ Res.; 78:262-273 (1996); Kaab etal., Circulation.; 98:1383-93 (1998)) which contribute to an adverseprognosis by predisposing to ventricular dysfunction and malignantarrhythmia. Significant morphological changes include increased rates ofapoptosis, (Haunstetter A and Izumo S, Circ Res.; 86:371-6 (2000))fibrosis, and chamber dilation.

The dichotomy between adaptive and maladaptive hypertrophy has beenappreciated for some time, and the mechanisms that determine howlong-standing hypertrophy ultimately progresses to overt heart failureare in the process of being elucidated. One biochemical hallmark of leftventricular hypertrophy induced by pressure overload is a shift inmyosin isoform from .alpha.- to .beta.-myosin heavy chains. (Delcayre Cand Swynghedauw B, Pflugers Arch.; 355:39-47 (1975)). This alteration inmyosin isoform expression result from transcriptionally mediatedalteration in gene expression. (Boehler et al., J Biol. Chem.;267:12979-12985 (1992)). Various lines of evidence suggest a decrease inthe expression of the sarcoplasmic reticulum Ca²⁺-cycling protein, Ca²⁺ATPase during the development of heart failure in several animal models,including humans with end-stage congestive heart failure, even though nochanges can be detected during the compensated hypertrophied stage.(Kiss et al., Circ Res.; 77:759-764 (1995); Feldman et al.,Circulation.; 75:331-9 (1987); Arai et al, Circ Res.; 72:463-469(1993)). These changes are associated with a decrease in sarcoplasmicreticulum Ca²⁺ transport. In addition, there are alterations in thelevel of phospholamban, sarcoplasmic reticulum Ca²⁺-release channels andin Ca²⁺ cycling proteins in the myofibrils and sarcolemma in differentanimal models with heart failure. (de la Bastie et al., Circ Res.;66:554-564 (1990); Mercadier et al., J Clin Invest.; 85:305-309 (1990)).These studies suggest that critical components of the Ca²⁺ cyclingsystem may be responsible, in part, for the transitions betweencompensated pressure-overload hypertrophy and congestive heart failure.

Hypertrophy that occurs as a consequence of pressure overload is termed“compensatory” on the premise that it facilitates ejection performanceby normalizing systolic wall stress. Recent experimental results,however, call into question the necessity of normalization of wallstress that results from hypertrophic growth of the heart. Thesefindings, largely from studies in genetically engineered mice, raise theprospect of modulating hypertrophic growth of the myocardium to affordclinical benefit without provoking hemodynamic compromise. (Frey et al.,supra, Dorn and Molkentin, supra; Frey et al., Circulation.; 109:1580-9(2004)).

It is generally accepted that cardiac hypertrophy can be adaptive insome situations, for example, in athletes. However, it is less clear ifa hypertrophic response to pathological situations, such as valvularheart disease, chronic arterial hypertension or a myocardial infarction,is initially a compensatory response and later becomes maladaptive or ifthis type of myocardial growth is detrimental from the outset.

It has been demonstrated that these different types of cardiachypertrophy differ both at the morphological as well as the molecularlevel. Exercise-induced cardiac hypertrophy is generally not accompaniedby an accumulation of collagen in the myocardium and usually does notexceed a modest increase in ventricular wall thickness. In addition,there are significantly differences in the expression levels for severalhypertrophic genes, such as BNP or ET-1. Further, the isoform expressionof α-/β-MHCs is regulated in opposite directions in exercise versuspressure overload-induced cardiac hypertrophy. However, somehypertrophic pathways, such as calcineurin-dependent signaling, appearto be activated in both pathological and physiological exercise-inducedhypertrophy, as demonstrated by the finding that the calcineurininhibitor can attenuate both phenotypes. Taken together, these dataindicate that exercise-associated (physiologic) versus pathologichypertrophy differ at the molecular level, but this does not exclude thepossibility that certain pathways may be involved in all phenotypes ofcardiac hypertrophy.

Since adult cardiomyocytes are terminally differentiated cells, many ofthe same intracellular signaling pathways that regulate proliferation incancer cells or immune cells instead regulate hypertrophic growth ofcardiomyocytes. The hypertrophic growth can be initiated by endocrine,paracrine, and autocrine factors that stimulate a wide array ofmembrane-bound receptors. Their activation results in the triggering ofmultiple cytoplasmic signal transduction cascades, which ultimatelyaffects nuclear factors and the regulation of gene expression. It haspreviously been documented that no single intracellular transductioncascade regulates cardiomyocyte hypertrophy in isolation, but insteadeach pathway operates as an integrated component between interdependentand cross-talking networks. Therefore, blockade of specificintracellular signaling pathways in the heart can dramatically affectthe entire hypertrophic response and effectively decrease cardiachypertrophy. Furthermore, specific activation of a number of discretesignal transduction pathways may be sufficient to activate the entirehypertrophic response through effects on other cross-talking signalingnetworks.

b. Valvular Heart Disease

The heart has four valves: the mitral valve (the only valve with twoflaps), the tricuspid, with three differently sized flaps, the aorticvalve, which opens to allow blood from the heart into the aorta, and thepulmonary valve. A number of disorders affecting the valves can resultin increased pressure in the chambers of the heart, which in turn canresult in cardiac hypertrophy. These conditions include mitral valvestenosis, mitral valve insufficiency, aortic valve insufficiency, aorticvalve stenosis, and tricuspid valve insufficiency. Several of theseconditions occur in persons who had undiagnosed or incompletely treatedrheumatic fever as a child. Rheumatic fever occurs most often inchildren who have a streptococcal throat infection (“strep throat”), andcan result in mitral stenosis, tricuspid stenosis, aortic insufficiency,aortic stenosis, multivalvular involvement or, less commonly, pulmonicstenosis. Unlike stenosis of blood vessels, which is typically caused bya build-up of lipids and cells on the interior of the vessel lumen,stenosis of heart valves is typically due to fusing of the flaps, to abuild-up of calcium on the flap, causing it to harden, to a congenitaldeformity, a weakening of valve tissue (“myxomatous degeneration”), oruse of certain medicines, such as fenfluramine and dexfenfluramine.

3. Preparation and Administration of Stem Cells

The administered stem cells will be appropriate to the tissue beingtargeted and treated or repaired. In varying embodiments, the stem cellsare multipotent or pluripotent, and can be isolated or induced. Invarying embodiments, the stem cells are mesenchymal stem cells, or stemcells derived or induced from the tissue of interest (e.g., muscletissue, particularly cardiac muscle tissue, nerve tissue). In varyingembodiments, the stem cells are isolated, derived or induced frommyocyte cells. In varying embodiments, the stem cells are isolated,derived or induced from cardiomyocyte cells. In varying embodiments, areisolated, derived or induced from cardiomyocytes or cardiac progenitorcells derived from multipotent stem cells, cardiomyocytes or cardiacprogenitor cells derived from pluripotent stem cells, cardiomyocytes orcardiac progenitor cells derived from induced pluripotent stem cells,adult cardiac progenitor cells or cardiac stem cells. Isolation,derivation and/or production of cardiac progenitor cells (Sca-1+/CD31−cells) and cardiac stem cells is known in the art, as published in,e.g., Wang, et al., PLoS One. 2014 Jun. 11; 9(6):e95247; Pagliari, etal., Front Physiol. 2014 Jun. 3; 5:210; Beltrami, et al., Cell. 2003Sep. 19; 114(6):763-76; Bolli, et al., Lancet. 2011 Nov. 26;378(9806):1847-57; Chugh, et al., Circulation. 2012 Sep. 11; 126(11Suppl 1):554-64; Makkar, et al., Lancet. 2012 Mar. 10;379(9819):895-904; Malliaras, et al., J Am Coll Cardiol. 2014 Jan. 21;63(2):110-22; Delewi, Heart. 2013 February; 99(4):225-32; Clifford, etal., Cochrane Database Syst Rev. 2012 Feb. 15; 2:CD006536; Dan, et al.,Am J Stem Cells. 2014 Mar. 13; 3(1):37-45; Wickham, et al., J BiomedMater Res B Appl Biomater. 2014 Mar. 24; and other references citedherein.

a. Mesenchymal Stem Cells

In varying embodiments, the stem cells are mesenchymal stem cells. Thebone marrow and adipose tissue of an adult mammal is a repository ofmesenchymal stem cells (MSCs). Bone marrow MSCs are self-renewing,clonal precursors of non-hematopoietic tissues. MSCs for use in thepresent methods can be isolated from a variety of tissues, includingbone marrow, muscle, fat (i.e., adipose), liver, dermis, gingiva andperiodontal ligament, using techniques known in the art. Depending onthe stimulus and the culture conditions employed, these cells can formbone, cartilage, tendon/ligament, muscle, marrow, adipose, and otherconnective tissues.

In some embodiments, the MSCs are derived from adipose tissue.Adipose-derived MSCs (AdMSCs) can be obtained from either autologous(self) and allogeneic (non-self) sources. The use of allogeneic MSCs inpatients is possible due to their low immunogenicity. However,autologous adMSCs are non-immunogeneic and considered to be safe inpeople and animals. AdMSCs can be administered either systematically(e.g., intravenous or intraarterially) or locally (e.g., directly tocardiac tissue, e.g., via the coronary artery) in the treatment ofdisorders. MSCs can be generated more efficiently and rapidly fromadipose tissue than from bone marrow. Fat- or adipose-derived MSCs arepresent in higher number and have a significantly higher proliferationrate then bone-marrow derived MSCs.

Generally, the MSCs useful for administration express on their cellsurface CD44, CD90 and CD105 and do not express on their cell surfaceCD4, CD34, CD45, CD80, CD86 or MHC-II. In various embodiments, the MSCsare adipose-derived mesenchymal stem cells (Ad-MSC). Ad-MSCs can becharacterized by the surface expression of CD5, CD44, CD90 (Thy-1) andCD105; and by the non-expression of CD3, CD4, CD18, CD34, CD45, CD49d,CD80, CD86 and MHC class II. In other embodiments, the MSCs are derivedfrom a non-adipose tissue, for example, bone marrow, liver, periodontalligament, gingiva and/or dermal tissues. In some embodiments, the MSCsare non-hematopoietic stem cells derived from bone marrow (e.g., do notexpress CD34 or CD45). Cell surface markers of cardiac progenitor cellscan include cKit, P-glycoprotein (a member of the multidrug resistanceprotein family), and Sca-1 (stem cell antigen 1), Sca-1-like, ISL LIMhomeobox 1 (ISL1), and/or kinase insert domain receptor (a type IIIreceptor tyrosine kinase) (KDR). See, e.g., Barile, et al., Nat ClinPract Cardiovasc Med. 2007 February; 4 Suppl 1:S9-S14.

As appropriate, the MSCs can be autologous (i.e., from the samesubject), syngeneic (i.e., from a subject having an identical or closelysimilar genetic makeup); allogeneic (i.e., from a subject of the samespecies) or xenogeneic to the subject (i.e., from a subject of adifferent species).

In various embodiments, the MSCs may be altered to enhance the viabilityof engrafted or transplanted cells. For example, the MSCs can beengineered to overexpress or to constitutively express Akt. See, e.g.,U.S. Patent Publication No. 2011/0091430.

b. Preparation

Tissue comprising mesenchymal stem cells (MSCs) is obtained andprocessed. As appropriate the tissue can be adipose, bone marrow,periodontal ligament, gingiva, muscle, liver or dermis. The obtainedMSCs can be autologous, syngeneic, allogeneic or xenogeneic to thesubject mammal. The tissue is processed to isolate the MSCs such thatthey can be cultured in vitro. Solid tissues can be minced and digestedwith a proteinase (e.g., collagenase), as appropriate. In varyingembodiments, the isolated MSCs can be cultured in vitro for at least 1passage, e.g., from about 2 passages to about 8 passages, e.g. for 2, 3,4, 5, 6, 7, 8 passages, as appropriate. At least 24 hours prior toinjection into the subject, e.g., 24, 36 or 48 hours prior to injection,the cultured MSCs are washed and then cultured in media comprising serumof the same species of the subject mammal (e.g., autologous, syngeneicand/or allogeneic serum) and in the absence of serum xenogeneic to thesubject mammal to wash out serum proteins allogeneic to the subjectmammal in order to avoid or minimize the risk of a potential transfusionreaction. For example, the present MSCs are substantially free of bovineand/or equine serum proteins prior to injection.

Prior to injection or engraftment into the subject mammal, the MSCs arerinsed and resuspended in a serum-free isotonic buffered solution (e.g.,phosphate-buffered saline, 0.9% sodium chloride, lactated Ringer'ssolution (LRS), Hank's Balanced Salt Solution (HBSS), Earle's BalancedSalt Solution (EBSS), etc.).

In varying embodiments, the MSCs administered to the subject are fresh(i.e., not frozen) and viable. In varying embodiments, the MSCs havebeen cultured in vitro for one or more passages prior to injection, sothe administered MSCs have never been frozen. The MSCs are evaluated forviability prior to administration. In varying embodiments, thepopulation of administered MSCs are at least about 50% viable, e.g., atleast about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 100%viable.

c. Administration of Stem Cells

The stem cells can be administered by any appropriate route. In variousembodiments, the stem cells are systemically administered, e.g.,intravenously, intra-arterially, or administered directly to the tissueof interest for treatment or repair.

In some embodiments, the stem cells are administered locally, e.g.,directly to cardiac tissue. As appropriate, the stem cells can beengrafted or transplanted into and/or around the tissue of interest,e.g., cardiac tissues. When engrafted or transplanted into and/or in thevicinity of one or more tissues of interest (e.g., cardiac tissues), thestem cells are administered within or within sufficient proximity ofinflamed or damaged lesions in tissue to mitigate and/or reverse ofdamage and/or destruction of the tissue. For example, the stem cells areengrafted or transplanted into or within sufficient proximity to thetissue of interest to prevent, reduce or inhibit damage and/ordestruction to the tissues.

As appropriate, injections of stem cells can be done after localanesthetics (e.g., lidocaine, bupivacaine) have been administered. It isalso possible to inject the stem cells in conjunction with localanesthetics added to the cell suspension. Injections can also be madewith the subject under general anesthesia with or without the use oflocal anesthetic agents (e.g., lidocaine).

In various embodiments, engraftment or transplantation of the stem cellscan be facilitated using a matrix or caged depot. For example, the stemcells can be engrafted or transplanted in a “caged cell” delivery devicewherein the cells are integrated into a biocompatible and/orbiologically inert matrix (e.g. a hydrogel or other polymer or anydevice) that restricts cell movement while allowing the cells to remainviable. Synthetic extracellular matrix and other biocompatible vehiclesfor delivery, retention, growth, and differentiation of stem cells areknown in the art and find use in the present methods. See, e.g.,Prestwich, J Control Release. 2011 Apr. 14, PMID 21513749; Perale, etal., Int J Artif Organs. (2011) 34(3):295-303; Suri, et al., Tissue EngPart A. (2010) 16(5):1703-16; Khetan, et al., J Vis Exp. (2009) Oct. 26;(32). pii: 1590; Salinas, et al., J Dent Res. (2009) 88(8):681-92;Schmidt, et al., J Biomed Mater Res A. (2008) 87(4):1113-22 and Xin, etal., Biomaterials (2007) 28:316-325.

As appropriate or desired, the engrafted or transplanted stem cells canbe modified to facilitate retention of the stem cells at the region ofinterest or the region of delivery. In other embodiments, the region ofinterest for engraftment or transplantation of the cells is modified inorder to facilitate retention of the stem cells at the region ofinterest or the region of delivery. In one embodiment, this can beaccomplished by introducing stromal cell derived factor-1 (SDF-1) intothe region of interest, e.g., using a linkage chemistry or integratedbiodegradable matrix (e.g., Poly(D,L-lactide-co-glycolide (PLGA) beads)that would provide a tunable temporal presence of the desired ligand upto several weeks. Stem cells bind to the immobilized SDF-1, therebyfacilitating the retention of stem cells that are delivered to theregion of interest for engraftment or transplantation. In otherembodiments, integrating cyclic arginine-glycine-aspartic acid peptideinto the region of interest can facilitate increased stem cell bindingand retention at the region of interest for engraftment ortransplantation. See, e.g., Ratliff, et al., Am J Pathol. (2010)177(2):873-83.

In some embodiments, at least about 1 million stem cells/kg subject areadministered or engrafted, e.g., at least about 2 million, 2.5 million,3 million, 3.5 million, 4 million, 5 million, 6 million, 7 million, 8million, 9 million or 10 million stem cells/kg subject are administered.In varying embodiments, about 1 million to about 10 million stemcells/kg subject, e.g., about 2 million to about 8 million stem cells/kgare administered or grafted.

In varying embodiments, at least about 5 million stem cells areadministered or engrafted, e.g., at least about 10 million, 15 million,20 million, 25 million, 30 million, 35 million, 40 million, 45 million,50 million, 55 million, 60 million, 65 million, 70 million, 75 million,80 million, 85 million, 90 million, 95 million or 100 million stem cellsare administered or engrafted. In varying embodiments, about 5 millionto about 100 million stem cells are administered or engrafted, e.g.,about 10 million to about 80 million stem cells are administered orengrafted. In varying embodiments, at least about 50 million stem cellsare administered or engrafted, e.g., at least about 100 million, 150million, 200 million, 250 million, 300 million, 350 million, 400million, 450 million, 500 million, 550 million, 600 million, 650million, 700 million, 750 million, 800 million, 850 million, 900million, 950 million or 1 billion stem cells are administered orengrafted.

In varying embodiments, the stem cells are administered, e.g.,intravenously, at a rate of about 1 million to about 10 million cellsper minute, e.g., at a rate of about 2 million to about 4 million cellsper minute, e.g., at a rate of about 2.5 million to about 3.5 millioncells per minute.

A regime of treatment or prevention may involve one or multipleinjections. For example, stem cells may be administered to the subject1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times, as appropriate. Subsequentadministrations of stem cells may be administered systemically orlocally. If administered locally, multiple injections of stem cells maybe administered to the same or different locations. Multiple injectionsof stem cells can be administered daily, weekly, bi-weekly, monthly,bi-monthly, every 3, 4, 5, or 6 months, or annually, or more or lessoften, as needed by the subject. The frequency of administration of thestem cells can change over a course of treatment, e.g., depending on howwell the engrafted or transplanted stem cells establish themselves atthe site of administration and the responsiveness of the subject. Thestem cells may be administered multiple times over a regime course ofseveral weeks, several months, several years, or for the remainder ofthe life of the subject, as needed or appropriate.

The total amount of cells that are envisioned for use depend upon thedesired effect, patient state, and the like, and may be determined byone skilled within the art. Dosages for any one patient depends uponmany factors, including the patient's species, size, body surface area,age, the particular stem cells to be administered, sex, scheduling androute of administration, general health, and other drugs beingadministered concurrently.

4. Agents that Increase the Production and/or Level of EpoxygenatedFatty Acids

Agents that increase epoxygenated fatty acids include epoxygenated fattyacids (e.g., including EETs), and inhibitors of soluble epoxidehydrolase (sEH).

a. Inhibitors of Soluble Epoxide Hydrolase (sEH)

Scores of sEH inhibitors are known, of a variety of chemical structures.Derivatives in which the urea, carbamate or amide pharmacophore areparticularly useful as sEH inhibitors. As used herein, “pharmacophore”refers to the section of the structure of a ligand that binds to thesEH. In various embodiments, the urea, carbamate or amide pharmacophoreis covalently bound to both an adamantane and to a 12 carbon chaindodecane. Derivatives that are metabolically stable are preferred, asthey are expected to have greater activity in vivo. Selective andcompetitive inhibition of sEH in vitro by a variety of urea, carbamate,and amide derivatives is taught, for example, by Morisseau et al., Proc.Natl. Acad. Sci. U.S.A, 96:8849-8854 (1999), which provides substantialguidance on designing urea derivatives that inhibit the enzyme.

Derivatives of urea are transition state mimetics that form a preferredgroup of sEH inhibitors. Within this group, N,N′-dodecyl-cyclohexyl urea(DCU), is preferred as an inhibitor, while N-cyclohexyl-N′-dodecylurea(CDU) is particularly preferred. Some compounds, such asdicyclohexylcarbodiimide (a lipophilic diimide), can decompose to anactive urea inhibitor such as DCU. Any particular urea derivative orother compound can be easily tested for its ability to inhibit sEH bystandard assays, such as those discussed herein. The production andtesting of urea and carbamate derivatives as sEH inhibitors is set forthin detail in, for example, Morisseau et al., Proc Natl Acad Sci (USA)96:8849-8854 (1999).

N-Adamantyl-N′-dodecyl urea (“ADU”) is both metabolically stable and hasparticularly high activity on sEH. (Both the 1- and the 2-admamantylureas have been tested and have about the same high activity as aninhibitor of sEH. Thus, isomers of adamantyl dodecyl urea are preferredinhibitors. It is further expected that N,N′-dodecyl-cyclohexyl urea(DCU), and other inhibitors of sEH, and particularly dodecanoic acidester derivatives of urea, are suitable for use in the methods.Preferred inhibitors include:

12-(3-Adamantan-1-yl-ureido)dodecanoic acid (AUDA),

12-(3-Adamantan-1-yl-ureido)dodecanoic acid butyl ester (AUDA-BE),

Adamantan-1-yl-3-{5-[2-(2-ethoxyethoxy)ethoxy]pentyl}urea (compound 950,also referred to herein as “AEPU”), and

Another preferred group of inhibitors are piperidines. The followingTables sets forth some exemplar inhibitors of sEH and their ability toinhibit sEH activity of the human enzyme and sEH from equine, ovine,porcine, feline and canine, expressed as the amount needed to reduce theactivity of the enzyme by 50% (expressed as “IC₅₀”).

TABLE 1 IC₅₀ values for selected alkylpiperidine-based sEH inhibitorsagainst human sEH

              n = 0     Compound  IC₅₀ (μM)^(a)               n = 1    Compound IC₅₀ (μM)^(a) R: H I 0.30 II 4.2 

3a 3.8  4.a 3.9 

3b 0.81 4b 2.6 

3c 1.2  4c 0.61

3d 0.01 4d 0.11 ^(a)As determined via a kinetic fluorescent assay.

TABLE 2 sEH inhibitors Structure Name sEHi #

3-(4-chlorophenyl)-1- (3,4-dichlorphenyl)urea or3,4,4′-trichlorocarbanilide  295 (TCC)

12-(3-adamantan- 1-yl-ureido) dodecanoic acid  700 (AUDA)

1-adamantanyl-3-{5- [2-(2- ethoxyethoxy)ethoxy]pentyl]}urea  950 (AEPU)

1-(1-acetypiperidin-4-yl)- 3-adamantanylurea 1153 (APAU)

trans-4-[4-(3-Adamantan- 1-yl-ureido)- cyclohexyloxy]- benzoic acid 1471(tAUCB)

1-trifluoromethoxyphenyl- 3-(1-acetylpiperidin-4-yl) urea 1555 (TPAU)

cis-4-[4-(3-Adamantan- 1-yl-ureido)- cyclohexyloxy]- benzoic acid 1686(cAUCB)

1-(1-methylsulfonyl- piperidin-4-yl)- 3-(4-trifluoromethoxy-phenyl)-urea 1709 (TUPS)

trans-4-{4-[3-(4- Trifluoromethoxy- phenyl)-ureido]- cyclohexyloxy}-benzoic acid 1728 (tTUCB)

1-trifluoromethoxyphenyl- 3-(1-propionylpiperidin- 4-yl) urea 1770(TPPU)

1-(1-ethylsulfonyl- piperidin-4-yl)-3- (4-trifluoromethoxy- phenyl)-urea2213 (TUPSE)

1-(1- (cyclopropanecarbonyl)piperidin- 4-yl)-3-(4-(trifluoromethoxy)phenyl)urea 2214 (CPTU)

trans-N-methyl-4- [4-(3-Adamantan-1- yl-ureido)- cyclohexyloxy]-benzamide 2225 (tMAUCB)

trans-N-methyl-4- [4-((3-trifluoromethyl- 4-chlorophenyl)-ureido)-cyclohexyloxy]- benzamide 2226 (tMTCUCB)

cis-N-methyl-4-{4- [3-(4-trifluoromethoxy- phenyl)-ureido]-cyclohexyloxy}-benzamide 2228 (cMTUCB)

1-cycloheptyl-3- (3-(1,5-diphenyl- 1H-pyrazol-3- yl)propyl)urea 2247(HDP3U)

A number of other sEH inhibitors which can be used in the methods andcompositions are set forth in co-owned applications PCT/US2013/024396,PCT/US2012/025074, PCT/US2011/064474, PCT/US2011/022901,PCT/US2008/072199, PCT/US2007/006412, PCT/US2005/038282,PCT/US2005/08765, PCT/US2004/010298 and U.S. Published PatentApplication Publication Nos: 2014/0088156, 2014/0038923, 2013/0274476,2013/0143925, 2013/0137726, 2011/0098322, 2005/0026844, each of which ishereby incorporated herein by reference in its entirety for allpurposes.

U.S. Pat. No. 5,955,496 (the '496 patent) also sets forth a number ofsEH inhibitors which can be used in the methods. One category of theseinhibitors comprises inhibitors that mimic the substrate for the enzyme.The lipid alkoxides (e.g., the 9-methoxide of stearic acid) are anexemplar of this group of inhibitors. In addition to the inhibitorsdiscussed in the '496 patent, a dozen or more lipid alkoxides have beentested as sEH inhibitors, including the methyl, ethyl, and propylalkoxides of oleic acid (also known as stearic acid alkoxides), linoleicacid, and arachidonic acid, and all have been found to act as inhibitorsof sEH.

In another group of embodiments, the '496 patent sets forth sEHinhibitors that provide alternate substrates for the enzyme that areturned over slowly. Exemplars of this category of inhibitors are phenylglycidols (e.g., S, S-4-nitrophenylglycidol), and chalcone oxides. The'496 patent notes that suitable chalcone oxides include 4-phenylchalconeoxide and 4-fluourochalcone oxide. The phenyl glycidols and chalconeoxides are believed to form stable acyl enzymes.

Additional inhibitors of sEH suitable for use in the methods are setforth in U.S. Pat. No. 6,150,415 (the '415 patent) and U.S. Pat. No.6,531,506 (the '506 patent). Two preferred classes of sEH inhibitors arecompounds of Formulas 1 and 2, as described in the '415 and '506patents. Means for preparing such compounds and assaying desiredcompounds for the ability to inhibit epoxide hydrolases are alsodescribed. The '506 patent, in particular, teaches scores of inhibitorsof Formula 1 and some twenty sEH inhibitors of Formula 2, which wereshown to inhibit human sEH at concentrations as low as 0.1 μM. Anyparticular sEH inhibitor can readily be tested to determine whether itwill work in the methods by standard assays. Esters and salts of thevarious compounds discussed above or in the cited patents, for example,can be readily tested by these assays for their use in the methods.

As noted above, chalcone oxides can serve as an alternate substrate forthe enzyme. While chalcone oxides have half-lives which depend in parton the particular structure, as a group the chalcone oxides tend to haverelatively short half-lives (a drug's half-life is usually defined asthe time for the concentration of the drug to drop to half its originalvalue. See, e.g., Thomas, G., Medicinal Chemistry: an introduction, JohnWiley & Sons Ltd. (West Sussex, England, 2000)). Since the various usescontemplate inhibition of sEH over differing periods of time which canbe measured in days, weeks, or months, chalcone oxides, and otherinhibitors which have a half-life whose duration is shorter than thepractitioner deems desirable, are preferably administered in a mannerwhich provides the agent over a period of time. For example, theinhibitor can be provided in materials that release the inhibitorslowly. Methods of administration that permit high local concentrationsof an inhibitor over a period of time are known, and are not limited touse with inhibitors which have short half-lives although, for inhibitorswith a relatively short half-life, they are a preferred method ofadministration.

In addition to the compounds in Formula 1 of the '506 patent, whichinteract with the enzyme in a reversible fashion based on the inhibitormimicking an enzyme-substrate transition state or reaction intermediate,one can have compounds that are irreversible inhibitors of the enzyme.The active structures such as those in the Tables or Formula 1 of the'506 patent can direct the inhibitor to the enzyme where a reactivefunctionality in the enzyme catalytic site can form a covalent bond withthe inhibitor. One group of molecules which could interact like thiswould have a leaving group such as a halogen or tosylate which could beattacked in an SN2 manner with a lysine or histidine. Alternatively, thereactive functionality could be an epoxide or Michael acceptor such asan α/β-unsaturated ester, aldehyde, ketone, ester, or nitrile.

Further, in addition to the Formula 1 compounds, active derivatives canbe designed for practicing the invention. For example, dicyclohexyl thiourea can be oxidized to dicyclohexylcarbodiimide which, with enzyme oraqueous acid (physiological saline), will form an activedicyclohexylurea. Alternatively, the acidic protons on carbamates orureas can be replaced with a variety of substituents which, uponoxidation, hydrolysis or attack by a nucleophile such as glutathione,will yield the corresponding parent structure. These materials are knownas prodrugs or protoxins (Gilman et al., The Pharmacological Basis ofTherapeutics, 7th Edition, MacMillan Publishing Company, New York, p. 16(1985)) Esters, for example, are common prodrugs which are released togive the corresponding alcohols and acids enzymatically (Yoshigae etal., Chirality, 9:661-666 (1997)). The drugs and prodrugs can be chiralfor greater specificity. These derivatives have been extensively used inmedicinal and agricultural chemistry to alter the pharmacologicalproperties of the compounds such as enhancing water solubility,improving formulation chemistry, altering tissue targeting, alteringvolume of distribution, and altering penetration. They also have beenused to alter toxicology profiles.

There are many prodrugs possible, but replacement of one or both of thetwo active hydrogens in the ureas described here or the single activehydrogen present in carbamates is particularly attractive. Suchderivatives have been extensively described by Fukuto and associates.These derivatives have been extensively described and are commonly usedin agricultural and medicinal chemistry to alter the pharmacologicalproperties of the compounds. (Black et al., Journal of Agricultural andFood Chemistry, 21(5):747-751 (1973); Fahmy et al, Journal ofAgricultural and Food Chemistry, 26(3):550-556 (1978); Jojima et al.,Journal of Agricultural and Food Chemistry, 31(3):613-620 (1983); andFahmy et al., Journal of Agricultural and Food Chemistry, 29(3):567-572(1981).)

Such active proinhibitor derivatives are within the scope of the presentinvention, and the just-cited references are incorporated herein byreference. Without being bound by theory, it is believed that suitableinhibitors mimic the enzyme transition state so that there is a stableinteraction with the enzyme catalytic site. The inhibitors appear toform hydrogen bonds with the nucleophilic carboxylic acid and apolarizing tyrosine of the catalytic site.

In some embodiments, the sEH inhibitor used in the methods taught hereinis a “soft drug.” Soft drugs are compounds of biological activity thatare rapidly inactivated by enzymes as they move from a chosen targetsite. EETs and simple biodegradable derivatives administered to an areaof interest may be considered to be soft drugs in that they are likelyto be enzymatically degraded by sEH as they diffuse away from the siteof interest following administration. Some sEHI, however, may diffuse orbe transported following administration to regions where their activityin inhibiting sEH may not be desired. Thus, multiple soft drugs fortreatment have been prepared. These include but are not limited tocarbamates, esters, carbonates and amides placed in the sEHI,approximately 7.5 angstroms from the carbonyl of the centralpharmacophore. These are highly active sEHI that yield biologicallyinactive metabolites by the action of esterase and/or amidase. Groupssuch as amides and carbamates on the central pharmacophores can also beused to increase solubility for applications in which that is desirablein forming a soft drug. Similarly, easily metabolized ethers maycontribute soft drug properties and also increase the solubility.

In some embodiments, sEH inhibition can include the reduction of theamount of sEH. As used herein, therefore, sEH inhibitors can thereforeencompass nucleic acids that inhibit expression of a gene encoding sEH.Many methods of reducing the expression of genes, such as reduction oftranscription and siRNA, are known, and are discussed in more detailbelow.

In various embodiments, a compound with combined functionality toconcurrently inhibit sEH and COX-2 is administered. Urea-containingpyrazoles that function as dual inhibitors of cyclooxygenase-2 andsoluble epoxide hydrolase are described, e.g., in Hwang, et al., J MedChem. (2011) 28; 54(8):3037-50.

Preferably, the inhibitor inhibits sEH without also significantlyinhibiting microsomal epoxide hydrolase (“mEH”). Preferably, atconcentrations of 100 μM, the inhibitor inhibits sEH activity by atleast 50% while not inhibiting mEH activity by more than 10%. Preferredcompounds have an IC₅₀ (inhibition potency or, by definition, theconcentration of inhibitor which reduces enzyme activity by 50%) of lessthan about 100 μM Inhibitors with IC₅₀s of less than 100 μM arepreferred, with IC₅₀s of less than 75 μM being more preferred and, inorder of increasing preference, an IC₅₀ of 50 μM, 40 μM, 30 μM, 25 μM,20 μM, 15 μM, 10 μM, 5 μM, 3 μM, 2 μM, 1 μM, 100 nM, 10 nM, 1.0 nM, oreven less, being still more preferred. Assays for determining sEHactivity are known in the art and described elsewhere herein. The IC₅₀determination of the inhibitor can be made with respect to an sEH enzymefrom the species subject to treatment (e.g., the subject receiving theinhibitor of sEH).

b. Cis-Epoxyeicosantrienoic Acids (“EETs”)

EETs, which are epoxides of arachidonic acid, are known to be effectorsof blood pressure, regulators of inflammation, and modulators ofvascular permeability. Hydrolysis of the epoxides by sEH diminishes thisactivity Inhibition of sEH raises the level of EETs since the rate atwhich the EETs are hydrolyzed into dihydroxyeicosatrienoic acids(“DHETs”) is reduced.

It has long been believed that EETs administered systemically would behydrolyzed too quickly by endogenous sEH to be helpful. For example, inone prior report of EETs administration, EETs were administered bycatheters inserted into mouse aortas. The EETs were infused continuouslyduring the course of the experiment because of concerns over the shorthalf-life of the EETs. See, Liao and Zeldin, International PublicationWO 01/10438 (hereafter “Liao and Zeldin”). It also was not known whetherendogenous sEH could be inhibited sufficiently in body tissues to permitadministration of exogenous EET to result in increased levels of EETsover those normally present. Further, it was thought that EETs, asepoxides, would be too labile to survive the storage and handlingnecessary for therapeutic use.

Studies from the laboratory of the present inventors, however, showedthat systemic administration of EETs in conjunction with inhibitors ofsEH had better results than did administration of sEH inhibitors alone.EETs were not administered by themselves in these studies since it wasanticipated they would be degraded too quickly to have a useful effect.Additional studies from the laboratory of the present inventors havesince shown, however, that administration of EETs by themselves has hadtherapeutic effect. Without wishing to be bound by theory, it issurmised that the exogenous EET overwhelms endogenous sEH, and allowsEETs levels to be increased for a sufficient period of time to havetherapeutic effect. Thus, EETs can be administered without alsoadministering an sEHI to provide a therapeutic effect. Moreover, EETs,if not exposed to acidic conditions or to sEH are stable and canwithstand reasonable storage, handling and administration.

In short, sEHI, EETs, or co-administration of sEHIs and of EETs, can beused in the methods of the present invention. In some embodiments, oneor more EETs are administered to the patient without also administeringan sEHI. In some embodiments, one or more EETs are administered shortlybefore or concurrently with administration of an sEH inhibitor to slowhydrolysis of the EET or EETs. In some embodiments, one or more EETs areadministered after administration of an sEH inhibitor, but before thelevel of the sEHI has diminished below a level effective to slow thehydrolysis of the EETs.

EETs useful in the methods of the present invention include 14,15-EET,8,9-EET and 11,12-EET, and 5,6 EETs. Preferably, the EETs areadministered as the methyl ester, which is more stable. Persons of skillwill recognize that the EETs are regioisomers, such as 8S,9R- and14R,15S-EET. 8,9-EET, 11,12-EET, and 14R,15S-EET, are commerciallyavailable from, for example, Sigma-Aldrich (catalog nos. E5516, E5641,and E5766, respectively, Sigma-Aldrich Corp., St. Louis, Mo.).

If desired, EETs, analogs, or derivatives that retain activity can beused in place of or in combination with unmodified EETs. Liao andZeldin, supra, define EET analogs as compounds with structuralsubstitutions or alterations in an EET, and include structural analogsin which one or more EET olefins are removed or replaced with acetyleneor cyclopropane groups, analogs in which the epoxide moiety is replacedwith oxitane or furan rings and heteroatom analogs. In other analogs,the epoxide moiety is replaced with ether, alkoxides, urea, amide,carbamate, difluorocycloprane, or carbonyl, while in others, thecarboxylic acid moiety is stabilized by blocking beta oxidation or isreplaced with a commonly used mimic, such as a nitrogen heterocycle, asulfonamide, or another polar functionality. In preferred forms, theanalogs or derivatives are relatively stable as compared to anunmodified EET because they are more resistant than an unmodified EET tosEH and to chemical breakdown. “Relatively stable” means the rate ofhydrolysis by sEH is at least 25% less than the hydrolysis of theunmodified EET in a hydrolysis assay, and more preferably 50% or morelower than the rate of hydrolysis of an unmodified EET. Liao and Zeldinshow, for example, episulfide and sulfonamide EETs derivatives. Amideand ester derivatives of EETs and that are relatively stable arepreferred embodiments. Whether or not a particular EET analog orderivative has the biological activity of the unmodified EET can bereadily determined by using it in standard assays, such as radio-ligandcompetition assays to measure binding to the relevant receptor. Asmentioned in the Definition section, above, for convenience ofreference, the term “EETs” as used herein refers to unmodified EETs, andEETs analogs and derivatives unless otherwise required by context.

In some embodiments, the EET or EETs are embedded or otherwise placed ina material that releases the EET over time. Materials suitable forpromoting the slow release of compositions such as EETs are known in theart. Optionally, one or more sEH inhibitors may also be placed in theslow release material.

Conveniently, the EET or EETs can be administered orally. Since EETs aresubject to degradation under acidic conditions, EETs intended for oraladministration can be coated with a coating resistant to dissolvingunder acidic conditions, but which dissolve under the mildly basicconditions present in the intestines. Suitable coatings, commonly knownas “enteric coatings” are widely used for products, such as aspirin,which cause gastric distress or which would undergo degradation uponexposure to gastric acid. By using coatings with an appropriatedissolution profile, the coated substance can be released in a chosensection of the intestinal tract. For example, a substance to be releasedin the colon is coated with a substance that dissolves at pH 6.5-7,while substances to be released in the duodenum can be coated with acoating that dissolves at pH values over 5.5. Such coatings arecommercially available from, for example, Rohm Specialty Acrylics (RohmAmerica LLC, Piscataway, N.J.) under the trade name “Eudragit®”. Thechoice of the particular enteric coating is not critical to thepractice.

c. Assays for Epoxide Hydrolase Activity

Any of a number of standard assays for determining epoxide hydrolaseactivity can be used to determine inhibition of sEH. For example,suitable assays are described in Gill, et al., Anal Biochem 131:273-282(1983); and Borhan, et al., Analytical Biochemistry 231:188-200 (1995)).Suitable in vitro assays are described in Zeldin et al., J Biol. Chem.268:6402-6407 (1993). Suitable in vivo assays are described in Zeldin etal., Arch Biochem Biophys 330:87-96 (1996). Assays for epoxide hydrolaseusing both putative natural substrates and surrogate substrates havebeen reviewed (see, Hammock, et al. In: Methods in Enzymology, VolumeIII, Steroids and Isoprenoids, Part B, (Law, J. H. and H. C. Rilling,eds. 1985), Academic Press, Orlando, Fla., pp. 303-311 and Wixtrom etal. , In: Biochemical Pharmacology and Toxicology, Vol. 1:Methodological Aspects of Drug Metabolizing Enzymes, (Zakim, D. and D.A. Vessey, eds. 1985), John Wiley & Sons, Inc., New York, pp. 1-93.Several spectral based assays exist based on the reactivity or tendencyof the resulting diol product to hydrogen bond (see, e.g., Wixtrom,supra, and Hammock. Anal. Biochem. 174:291-299 (1985) and Dietze, et al.Anal. Biochem. 216:176-187 (1994)).

The enzyme also can be detected based on the binding of specific ligandsto the catalytic site which either immobilize the enzyme or label itwith a probe such as dansyl, fluoracein, luciferase, green fluorescentprotein or other reagent. The enzyme can be assayed by its hydration ofEETs, its hydrolysis of an epoxide to give a colored product asdescribed by Dietze et al., 1994, supra, or its hydrolysis of aradioactive surrogate substrate (Borhan et al., 1995, supra). The enzymealso can be detected based on the generation of fluorescent productsfollowing the hydrolysis of the epoxide. Numerous methods of epoxidehydrolase detection have been described (see, e.g., Wixtrom, supra).

The assays are normally carried out with a recombinant enzyme followingaffinity purification. They can be carried out in crude tissuehomogenates, cell culture or even in vivo, as known in the art anddescribed in the references cited above.

d. Other Means of Inhibiting sEH Activity

Other means of inhibiting sEH activity or gene expression can also beused in the methods. For example, a nucleic acid molecule complementaryto at least a portion of the human sEH gene can be used to inhibit sEHgene expression. Means for inhibiting gene expression using short RNAmolecules, for example, are known. Among these are short interfering RNA(siRNA), small temporal RNAs (stRNAs), and micro-RNAs (miRNAs). Shortinterfering RNAs silence genes through a mRNA degradation pathway, whilestRNAs and miRNAs are approximately 21 or 22 nt RNAs that are processedfrom endogenously encoded hairpin-structured precursors, and function tosilence genes via translational repression. See, e.g., McManus et al.,RNA, 8(6):842-50 (2002); Morris et al., Science, 305(5688):1289-92(2004); He and Hannon, Nat Rev Genet. 5(7):522-31 (2004).

“RNA interference,” a form of post-transcriptional gene silencing(“PTGS”), describes effects that result from the introduction ofdouble-stranded RNA into cells (reviewed in Fire, A. Trends Genet15:358-363 (1999); Sharp, P. Genes Dev 13:139-141 (1999); Hunter, C.Curr Biol 9:R440-R442 (1999); Baulcombe. D. Curr Biol 9:R599-R601(1999); Vaucheret et al. Plant J 16: 651-659 (1998)). RNA interference,commonly referred to as RNAi, offers a way of specifically inactivatinga cloned gene, and is a powerful tool for investigating gene function.

The active agent in RNAi is a long double-stranded (antiparallel duplex)RNA, with one of the strands corresponding or complementary to the RNAwhich is to be inhibited. The inhibited RNA is the target RNA. The longdouble stranded RNA is chopped into smaller duplexes of approximately 20to 25 nucleotide pairs, after which the mechanism by which the smallerRNAs inhibit expression of the target is largely unknown at this time.While RNAi was shown initially to work well in lower eukaryotes, formammalian cells, it was thought that RNAi might be suitable only forstudies on the oocyte and the preimplantation embryo.

In mammalian cells other than these, however, longer RNA duplexesprovoked a response known as “sequence non-specific RNA interference,”characterized by the non-specific inhibition of protein synthesis.

Further studies showed this effect to be induced by dsRNA of greaterthan about 30 base pairs, apparently due to an interferon response. Itis thought that dsRNA of greater than about 30 base pairs binds andactivates the protein PKR and 2′,5′-oligonucleotide synthetase(2′,5′-AS). Activated PKR stalls translation by phosphorylation of thetranslation initiation factors eIF2α, and activated 2′,5′-AS causes mRNAdegradation by 2′,5′-oligonucleotide-activated ribonuclease L. Theseresponses are intrinsically sequence-nonspecific to the inducing dsRNA;they also frequently result in apoptosis, or cell death. Thus, mostsomatic mammalian cells undergo apoptosis when exposed to theconcentrations of dsRNA that induce RNAi in lower eukaryotic cells.

More recently, it was shown that RNAi would work in human cells if theRNA strands were provided as pre-sized duplexes of about 19 nucleotidepairs, and RNAi worked particularly well with small unpaired 3′extensions on the end of each strand (Elbashir et al. Nature 411:494-498 (2001)). In this report, siRNA were applied to cultured cells bytransfection in oligofectamine micelles. These RNA duplexes were tooshort to elicit sequence-nonspecific responses like apoptosis, yet theyefficiently initiated RNAi. Many laboratories then tested the use ofsiRNA to knock out target genes in mammalian cells. The resultsdemonstrated that siRNA works quite well in most instances.

For purposes of reducing the activity of sEH, siRNAs to the geneencoding sEH can be specifically designed using computer programs. Thecloning, sequence, and accession numbers of the human sEH sequence areset forth in Beetham et al., Arch. Biochem. Biophys. 305(1):197-201(1993). An exemplary amino acid sequence of human sEH (GenBank AccessionNo. L05779; SEQ ID NO:1) and an exemplary nucleotide sequence encodingthat amino acid sequence (GenBank Accession No. AAA02756; SEQ ID NO:2)are set forth in U.S. Pat. No. 5,445,956. The nucleic acid sequence ofhuman sEH is also published as GenBank Accession No. NM_(—)001979.4; theamino acid sequence of human sEH is also published as GenBank AccessionNo. NP_(—)001970.2.

A program, siDESIGN from Dharmacon, Inc. (Lafayette, Colo.), permitspredicting siRNAs for any nucleic acid sequence, and is available on theWorld Wide Web at dharmacon.com. Programs for designing siRNAs are alsoavailable from others, including Genscript (available on the Web atgenscript.com/ssl-bin/app/rnai) and, to academic and non-profitresearchers, from the Whitehead Institute for Biomedical Research foundon the worldwide web at“jura.wi.mit.edu/pubint/http://iona.wi.mit.edu/siRNAext/.”

For example, using the program available from the Whitehead Institute,the following sEH target sequences and siRNA sequences can be generated:

1) Target: (SEQ ID NO: 3) CAGTGTTCATTGGCCATGACTGG Sense-siRNA:(SEQ ID NO: 4) 5′-GUGUUCAUUGGCCAUGACUTT-3′ Antisense-siRNA:(SEQ ID NO: 5) 5′-AGUCAUGGCCAAUGAACACTT-3′ 2) Target: (SEQ ID NO: 6)GAAAGGCTATGGAGAGTCATCTG Sense-siRNA: (SEQ ID NO: 7)5′-AAGGCUAUGGAGAGUCAUCTT-3′ Antisense-siRNA: (SEQ ID NO: 8)5′-GAUGACUCUCCAUAGCCUUTT-3′ 3) Target (SEQ ID NO: 9)AAAGGCTATGGAGAGTCATCTGC Sense-siRNA: (SEQ ID NO: 10)5′-AGGCUAUGGAGAGUCAUCUTT-3′ Antisense-siRNA: (SEQ ID NO: 11)5′-AGAUGACUCUCCAUAGCCUTT-3′ 4) Target: (SEQ ID NO: 12)CAAGCAGTGTTCATTGGCCATGA Sense-siRNA: (SEQ ID NO: 13)5′-AGCAGUGUUCAUUGGCCAUTT-3′ Antisense-siRNA: (SEQ ID NO: 14)5′-AUGGCCAAUGAACACUGCUTT-3′ 5) Target: (SEQ ID NO: 15)CAGCACATGGAGGACTGGATTCC Sense-siRNA: (SEQ ID NO: 16)5′-GCACAUGGAGGACUGGAUUTT-3′ Antisense-siRNA: (SEQ ID NO: 17)5′-AAUCCAGUCCUCCAUGUGCTT-3′

Alternatively, siRNA can be generated using kits which generate siRNAfrom the gene. For example, the “Dicer siRNA Generation” kit (catalognumber T510001, Gene Therapy Systems, Inc., San Diego, Calif.) uses therecombinant human enzyme “dicer” in vitro to cleave long double strandedRNA into 22 by siRNAs. By having a mixture of siRNAs, the kit permits ahigh degree of success in generating siRNAs that will reduce expressionof the target gene. Similarly, the Silencer™ siRNA Cocktail Kit (RNaseIII) (catalog no. 1625, Ambion, Inc., Austin, Tex.) generates a mixtureof siRNAs from dsRNA using RNase III instead of dicer. Like dicer, RNaseIII cleaves dsRNA into 12-30 by dsRNA fragments with 2 to 3 nucleotide3′ overhangs, and 5′-phosphate and 3′-hydroxyl termini. According to themanufacturer, dsRNA is produced using T7 RNA polymerase, and reactionand purification components included in the kit. The dsRNA is thendigested by RNase III to create a population of siRNAs. The kit includesreagents to synthesize long dsRNAs by in vitro transcription and todigest those dsRNAs into siRNA-like molecules using RNase III. Themanufacturer indicates that the user need only supply a DNA templatewith opposing T7 phage polymerase promoters or two separate templateswith promoters on opposite ends of the region to be transcribed.

The siRNAs can also be expressed from vectors. Typically, such vectorsare administered in conjunction with a second vector encoding thecorresponding complementary strand. Once expressed, the two strandsanneal to each other and form the functional double stranded siRNA. Oneexemplar vector suitable for use in the invention is pSuper, availablefrom OligoEngine, Inc. (Seattle, Wash.). In some embodiments, the vectorcontains two promoters, one positioned downstream of the first and inantiparallel orientation. The first promoter is transcribed in onedirection, and the second in the direction antiparallel to the first,resulting in expression of the complementary strands. In yet another setof embodiments, the promoter is followed by a first segment encoding thefirst strand, and a second segment encoding the second strand. Thesecond strand is complementary to the palindrome of the first strand.Between the first and the second strands is a section of RNA serving asa linker (sometimes called a “spacer”) to permit the second strand tobend around and anneal to the first strand, in a configuration known asa “hairpin.”

The formation of hairpin RNAs, including use of linker sections, is wellknown in the art. Typically, an siRNA expression cassette is employed,using a Polymerase III promoter such as human U6, mouse U6, or human Hl.The coding sequence is typically a 19-nucleotide sense siRNA sequencelinked to its reverse complementary antisense siRNA sequence by a shortspacer. Nine-nucleotide spacers are typical, although other spacers canbe designed. For example, the Ambion website indicates that itsscientists have had success with the spacer TTCAAGAGA (SEQ ID NO:18).Further, 5-6 T's are often added to the 3′ end of the oligonucleotide toserve as a termination site for Polymerase III. See also, Yu et al., MolTher 7(2):228-36 (2003); Matsukura et al., Nucleic Acids Res 31(15):e77(2003).

As an example, the siRNA targets identified above can be targeted byhairpin siRNA as follows. To attack the same targets by short hairpinRNAs, produced by a vector (permanent RNAi effect), sense and antisensestrand can be put in a row with a loop forming sequence in between andsuitable sequences for an adequate expression vector to both ends of thesequence. The following are non-limiting examples of hairpin sequencesthat can be cloned into the pSuper vector:

1) Target: (SEQ ID NO: 19) CAGTGTTCATTGGCCATGACTGG Sense strand:(SEQ ID NO: 20) 5′-GATCCCCGTGTTCATTGGCCATGACTTTCAAGAGAAGTCATGGCCAATGAACACTTTTT-3′ Antisense strand: (SEQ ID NO: 21)5′-AGCTAAAAAGTGTTCATTGGCCATGACTTCTCTTGAAAGT CATGGCCAATGAACACGGG-3′ 2)Target: (SEQ ID NO: 22) GAAAGGCTATGGAGAGTCATCTG Sense strand:(SEQ ID NO: 23) 5′-GATCCCCAAGGCTATGGAGAGTCATCTTCAAGAGAGATGACTCTCCATAGCCTTTTTTT-3′ Antisense strand: (SEQ ID NO: 24)5′-AGCTAAAAAAAGGCTATGGAGAGTCATCTCTCTTGAAGAT GACTCTCCATAGCCTTGGG-3′ 3)Target: (SEQ ID NO: 25) AAAGGCTATGGAGAGTCATCTGC Sense strand:(SEQ ID NO: 26) 5′-GATCCCCAGGCTATGGAGAGTCATCTTTCAAGAGAAGATGACTCTCCATAGCCTTTTTT-3′ Antisense strand: (SEQ ID NO: 27)5′-AGCTAAAAAAGGCTATGGAGAGTCATCATCTCTTGAAAGA TGACTCTCCATAGCCTGGG-3′ 4)Target: (SEQ ID NO: 28) CAAGCAGTGTTCATTGGCCATGA Sense strand:(SEQ ID NO: 29) 5′-GATCCCCAGCAGTGTTCATTGGCCATTTCAAGAGAATGGCCAATGAACACTGCTTTTTT-3′ Antisense strand: (SEQ ID NO: 30)5′-AGCTAAAAAAGCAGTGTTCATTGGCCATTCTCTTGAAATG GCCAATGAACACTGCTGGG-3′ 5)Target: (SEQ ID NO: 31) CAGCACATGGAGGACTGGATTCC Sense strand(SEQ ID NO: 32) 5′-GATCCCCGCACATGGAGGACTGGATTTTCAAGAGAAATCCAGTCCTCCATGTGCTTTTT-3′ Antisense strand: (SEQ ID NO: 33)5′-AGCTAAAAAGCACATGGAGGACTGGATTTCTCTTGAAAAT CCAGTCCTCCATGTGCGGG-3′

In addition to siRNAs, other means are known in the art for inhibitingthe expression of antisense molecules, ribozymes, and the like are wellknown to those of skill in the art. The nucleic acid molecule can be aDNA probe, a riboprobe, a peptide nucleic acid probe, a phosphorothioateprobe, or a 2′-O methyl probe.

Generally, to assure specific hybridization, the antisense sequence issubstantially complementary to the target sequence. In certainembodiments, the antisense sequence is exactly complementary to thetarget sequence. The antisense polynucleotides may also include,however, nucleotide substitutions, additions, deletions, transitions,transpositions, or modifications, or other nucleic acid sequences ornon-nucleic acid moieties so long as specific binding to the relevanttarget sequence corresponding to the sEH gene is retained as afunctional property of the polynucleotide. In one embodiment, theantisense molecules form a triple helix-containing, or “triplex” nucleicacid. Triple helix formation results in inhibition of gene expressionby, for example, preventing transcription of the target gene (see, e.g.,Cheng et al., 1988, J. Biol. Chem. 263:15110; Ferrin and Camerini-Otero,1991, Science 354:1494; Ramdas et al., 1989, J. Biol. Chem. 264:17395;Strobel et al., 1991, Science 254:1639; and Rigas et al., 1986, Proc.Natl. Acad. Sci. U.S.A. 83:9591)

Antisense molecules can be designed by methods known in the art. Forexample, Integrated DNA Technologies (Coralville, Iowa) makes availablea program found on the worldwide web“biotools.idtdna.com/antisense/AntiSense.aspx”, which will provideappropriate antisense sequences for nucleic acid sequences up to 10,000nucleotides in length. Using this program with the sEH gene provides thefollowing exemplar sequences:

1) (SEQ ID NO: 34) UGUCCAGUGCCCACAGUCCU 2) (SEQ ID NO: 35)UUCCCACCUGACACGACUCU 3) (SEQ ID NO: 36) GUUCAGCCUCAGCCACUCCU 4)(SEQ ID NO: 37) AGUCCUCCCGCUUCACAGA 5) (SEQ ID NO: 38)GCCCACUUCCAGUUCCUUUCC

In another embodiment, ribozymes can be designed to cleave the mRNA at adesired position. (See, e.g., Cech, 1995, Biotechnology 13:323; andEdgington, 1992, Biotechnology 10:256 and Hu et al., PCT Publication WO94/03596).

The antisense nucleic acids (DNA, RNA, modified, analogues, and thelike) can be made using any suitable method for producing a nucleicacid, such as the chemical synthesis and recombinant methods disclosedherein and known to one of skill in the art. In one embodiment, forexample, antisense RNA molecules may be prepared by de novo chemicalsynthesis or by cloning. For example, an antisense RNA can be made byinserting (ligating) a sEH gene sequence in reverse orientation operablylinked to a promoter in a vector (e.g., plasmid). Provided that thepromoter and, preferably termination and polyadenylation signals, areproperly positioned, the strand of the inserted sequence correspondingto the noncoding strand are transcribed and act as an antisenseoligonucleotide.

It are appreciated that the oligonucleotides can be made usingnonstandard bases (e.g., other than adenine, cytidine, guanine, thymine,and uridine) or nonstandard backbone structures to provides desirableproperties (e.g., increased nuclease-resistance, tighter-binding,stability or a desired Tm). Techniques for rendering oligonucleotidesnuclease-resistant include those described in PCT Publication WO94/12633. A wide variety of useful modified oligonucleotides may beproduced, including oligonucleotides having a peptide-nucleic acid (PNA)backbone (Nielsen et al., 1991, Science 254:1497) or incorporating2′-O-methyl ribonucleotides, phosphorothioate nucleotides, methylphosphonate nucleotides, phosphotriester nucleotides, phosphorothioatenucleotides, phosphoramidates.

Proteins have been described that have the ability to translocatedesired nucleic acids across a cell membrane. Typically, such proteinshave amphiphilic or hydrophobic subsequences that have the ability toact as membrane-translocating carriers. For example, homeodomainproteins have the ability to translocate across cell membranes. Theshortest internalizable peptide of a homeodomain protein, Antennapedia,was found to be the third helix of the protein, from amino acid position43 to 58 (see, e.g., Prochiantz, Current Opinion in Neurobiology6:629-634 (1996). Another subsequence, the h (hydrophobic) domain ofsignal peptides, was found to have similar cell membrane translocationcharacteristics (see, e.g., Lin et al., J. Biol. Chem. 270:14255-14258(1995)). Such subsequences can be used to translocate oligonucleotidesacross a cell membrane. Oligonucleotides can be conveniently derivatizedwith such sequences. For example, a linker can be used to link theoligonucleotides and the translocation sequence. Any suitable linker canbe used, e.g., a peptide linker or any other suitable chemical linker.

More recently, it has been discovered that siRNAs can be introduced intomammals without eliciting an immune response by encapsulating them innanoparticles of cyclodextrin. Information on this method can be foundon the worldwide web at“nature.com/news/2005/050418/full/050418-6.html.”

In another method, the nucleic acid is introduced directly intosuperficial layers of the skin or into muscle cells by a jet ofcompressed gas or the like. Methods for administering nakedpolynucleotides are well known and are taught, for example, in U.S. Pat.No. 5,830,877 and International Publication Nos. WO 99/52483 and94/21797. Devices for accelerating particles into body tissues usingcompressed gases are described in, for example, U.S. Pat. Nos.6,592,545, 6,475,181, and 6,328,714. The nucleic acid may be lyophilizedand may be complexed, for example, with polysaccharides to form aparticle of appropriate size and mass for acceleration into tissue.Conveniently, the nucleic acid can be placed on a gold bead or otherparticle which provides suitable mass or other characteristics. Use ofgold beads to carry nucleic acids into body tissues is taught in, forexample, U.S. Pat. Nos. 4,945,050 and 6,194,389.

The nucleic acid can also be introduced into the body in a virusmodified to serve as a vehicle without causing pathogenicity. The viruscan be, for example, adenovirus, fowlpox virus or vaccinia virus.

miRNAs and siRNAs differ in several ways: miRNA derive from points inthe genome different from previously recognized genes, while siRNAsderive from mRNA, viruses or transposons, miRNA derives from hairpinstructures, while siRNA derives from longer duplexed RNA, miRNA isconserved among related organisms, while siRNA usually is not, and miRNAsilences loci other than that from which it derives, while siRNAsilences the loci from which it arises. Interestingly, miRNAs tend notto exhibit perfect complementarity to the mRNA whose expression theyinhibit. See, McManus et al., supra. See also, Cheng et al., NucleicAcids Res. 33(4):1290-7 (2005); Robins and Padgett, Proc Natl Acad SciUSA. 102(11):4006-9 (2005); Brennecke et al., PLoS Biol. 3(3):e85(2005). Methods of designing miRNAs are known. See, e.g., Zeng et al.,Methods Enzymol. 392:371-80 (2005); Krol et al., J Biol Chem.279(40):42230-9 (2004); Ying and Lin, Biochem Biophys Res Commun.326(3):515-20 (2005).

e. Epoxygenated Fatty Acids

In some embodiments, an epoxygenated fatty acid is administered as anagent that increases epoxygenated fatty acids. Illustrative epoxygenatedfatty acids include epoxides of linoleic acid, eicosapentaenoic acid(“EPA”) and docosahexaenoic acid (“DHA”).

The fatty acids eicosapentaenoic acid (“EPA”) and docosahexaenoic acid(“DHA”) have recently become recognized as having beneficial effects,and fish oil tablets, which are a good source of these fatty acids, arewidely sold as supplements. In 2003, it was reported that these fattyacids reduced pain and inflammation. Sethi, S. et al., Blood 100:1340-1346 (2002). The paper did not identify the mechanism of action,nor the agents responsible for this relief

Cytochrome P450 (“CYP450”) metabolism produces cis-epoxydocosapentaenoicacids (“EpDPEs”) and cis-epoxyeicosatetraenoic acids (“EpETEs”) fromdocosahexaenoic acid (“DHA”) and eicosapentaenoic acid (“EPA”),respectively. These epoxides are known endothelium-derivedhyperpolarizing factors (“EDHFs”). These EDHFs, and others yetunidentified, are mediators released from vascular endothelial cells inresponse to acetylcholine and bradykinin, and are distinct from the NOS-(nitric oxide) and COX-derived (prostacyclin) vasodilators. Overallcytochrome P450 (CYP450) metabolism of polyunsaturated fatty acidsproduces epoxides, such as EETs, which are prime candidates for theactive mediator(s). 14(15)-EpETE, for example, is derived viaepoxidation of the 14,15-double bond of EPA and is the ω-3 homolog of14(15)-EpETrE (“14(15)EET”) derived via epoxidation of the 14,15-doublebond of arachidonic acid.

As mentioned, it is beneficial to elevate the levels of EETs, which areepoxides of the fatty acid arachidonic acid. Our studies of the effectsof EETs has led us to realization that the anti-inflammatory effect ofEPA and DHA are likely due to increasing the levels of the epoxides ofthese two fatty acids. Thus, increasing the levels of epoxides of EPA,of DHA, or of both, will act to reduce pain and inflammation, andsymptoms associated with diabetes and metabolic syndromes, in mammals inneed thereof. This beneficial effect of the epoxides of these fattyacids has not been previously recognized. Moreover, these epoxides havenot previously been administered as agents, in part because, as notedabove, epoxides have generally been considered too labile to beadministered.

Like EETs, the epoxides of EPA and DHA are substrates for sEH. Theepoxides of EPA and DHA are produced in the body at low levels by theaction of cytochrome P450s. Endogenous levels of these epoxides can bemaintained or increased by the administration of sEHI. However, theendogeous production of these epoxides is low and usually occurs inrelatively special circumstances, such as the resolution ofinflammation. Our expectation is that administering these epoxides fromexogenous sources will aid in the resolution of inflammation and inreducing pain, as well as with symptoms of diabetes and metabolicsyndromes. It is further beneficial with pain or inflammation to inhibitsEH with sEHI to reduce hydrolysis of these epoxides, therebymaintaining them at relatively high levels.

EPA has five unsaturated bonds, and thus five positions at whichepoxides can be formed, while DHA has six. The epoxides of EPA aretypically abbreviated and referred to generically as “EpETEs”, while theepoxides of DHA are typically abbreviated and referred to generically as“EpDPEs”. The specific regioisomers of the epoxides of each fatty acidare set forth in the following Table:

TABLE 3 Regioisomers of Eicosapentaenoic acid (“EPA”) epoxides: 1.Formal name: (±)5(6)-epoxy-8Z,11Z,14Z,17Z- eicosatetraenoic acid,Synonym 5(6)-epoxy Eicosatetraenoic acid Abbreviation 5(6)-EpETE 2.Formal name: (±)8(9)-epoxy-5Z,11Z,14Z,17Z- eicosatetraenoic acid,Synonym 8(9)-epoxy Eicosatetraenoic acid Abbreviation 8(9)-EpETE 3.Formal name: (±)11(12)-epoxy-5Z,8Z,14Z,17Z- eicosatetraenoic acid,Synonym 11(12)-epoxy Eicosatetraenoic acid Abbreviation 11(12)-EpETE 4.Formal name: (±)14(15)-epoxy-5Z,8Z,11Z,17Z- eicosatetraenoic acid,Synonym 14(15)-epoxy Eicosatetraenoic acid Abbreviation 14(15)-EpETE 5.Formal name: (±)17(18)-epoxy-5Z,8Z,11Z,14Z- eicosatetraenoic acid,Synonym 17(18)-epoxy Eicosatetraenoic acid Abbreviation 17(18)-EpETERegioisomers of Docosahexaenoic acid (“DHA”) epoxides: 1. Formal name:(±) 4(5)-epoxy-7Z,10Z,13Z,16Z,19Z- docosapentaenoic acid, Synonym4(5)-epoxy Docosapentaenoic acid Abbreviation 4(5)-EpDPE 2. Formal name:(±) 7(8)-epoxy-4Z,10Z,13Z,16Z,19Z- docosapentaenoic acid, Synonym7(8)-epoxy Docosapentaenoic acid Abbreviation 7(8)-EpDPE 3. Formal name:(±)10(11)-epoxy-4Z,7Z,13Z,16Z,19Z- docosapentaenoic acid, Synonym10(11)-epoxy Docosapentaenoic acid Abbreviation 10(11)-EpDPE 4. Formalname: (±)13(14)-epoxy-4Z,7Z,10Z,16Z,19Z- docosapentaenoic acid, Synonym13(14)-epoxy Docosapentaenoic acid Abbreviation 13(14)-EpDPE 5. Formalname: (±) 16(17)-epoxy-4Z,7Z,10Z,13Z,19Z- docosapentaenoic acid, Synonym16(17)-epoxy Docosapentaenoic acid Abbreviation 16(17)-EpDPE 6. Formalname: (±) 19(20)-epoxy-4Z,7Z,10Z,13Z,16Z- docosapentaenoic acid, Synonym19(20)-epoxy Docosapentaenoic acid Abbreviation 19(20)-EpDPE

Any of these epoxides, or combinations of any of these, can beadministered in the compositions and methods.

5. Formulation and Administration

In various embodiments of the compositions, the agent that increasesepoxygenated fatty acids (e.g., an inhibitor of sEH, an EET, anepoxygenated fatty acid, and mixtures thereof) is co-administered withthe population of stem cells. In some embodiments, the agent thatincreases epoxygenated fatty acids comprises an epoxide of EPA, anepoxide of DHA, or epoxides of both, and an sEHI.

The agent that increases epoxygenated fatty acids can be prepared andadministered in a wide variety of oral, parenteral and topical dosageforms. In varying embodiments, the agent that increases epoxygenatedfatty acids can be administered orally, by injection, that is,intravenously, intramuscularly, intracutaneously, subcutaneously,intraduodenally, or intraperitoneally. The agent that increasesepoxygenated fatty acids can also be administered by inhalation, forexample, intranasally. Additionally, the agent that increasesepoxygenated fatty acids can be administered transdermally. Accordingly,in some embodiments, the methods contemplate administration ofcompositions comprising a pharmaceutically acceptable carrier orexcipient, an agent that increases epoxygenated fatty acids (e.g., ansEHI or a pharmaceutically acceptable salt of the inhibitor and,optionally, one or more EETs or epoxides of EPA or of DHA, or of both),and optionally an anti-inflammatory agent. In some embodiments, themethods comprise administration of an sEHI and one or more epoxides ofEPA or of DHA, or of both.

For preparing the pharmaceutical compositions, the pharmaceuticallyacceptable carriers can be either solid or liquid. Solid formpreparations include powders, tablets, pills, capsules, cachets,suppositories, and dispersible granules. A solid carrier can be one ormore substances which may also act as diluents, flavoring agents,binders, preservatives, tablet disintegrating agents, or anencapsulating material.

In powders, the carrier is a finely divided solid which is in a mixturewith the finely divided active component. In tablets, the activecomponent is mixed with the carrier having the necessary bindingproperties in suitable proportions and compacted in the shape and sizedesired. The powders and tablets preferably contain from 5% or 10% to70% of the active compound. Suitable carriers are magnesium carbonate,magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch,gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, alow melting wax, cocoa butter, and the like. The term “preparation” isintended to include the formulation of the active compound withencapsulating material as a carrier providing a capsule in which theactive component with or without other carriers, is surrounded by acarrier, which is thus in association with it. Similarly, cachets andlozenges are included. Tablets, powders, capsules, pills, cachets, andlozenges can be used as solid dosage forms suitable for oraladministration.

For preparing suppositories, a low melting wax, such as a mixture offatty acid glycerides or cocoa butter, is first melted and the activecomponent is dispersed homogeneously therein, as by stirring. The moltenhomogeneous mixture is then poured into convenient sized molds, allowedto cool, and thereby to solidify.

Liquid form preparations include solutions, suspensions, and emulsions,for example, water or water/propylene glycol solutions. For parenteralinjection, liquid preparations can be formulated in solution in aqueouspolyethylene glycol solution. Transdermal administration can beperformed using suitable carriers. If desired, apparatuses designed tofacilitate transdermal delivery can be employed. Suitable carriers andapparatuses are well known in the art, as exemplified by U.S. Pat. Nos.6,635,274, 6,623,457, 6,562,004, and 6,274,166.

Aqueous solutions suitable for oral use can be prepared by dissolvingthe active component in water and adding suitable colorants, flavors,stabilizers, and thickening agents as desired. Aqueous suspensionssuitable for oral use can be made by dispersing the finely dividedactive components in water with viscous material, such as natural orsynthetic gums, resins, methylcellulose, sodium carboxymethylcellulose,and other well-known suspending agents.

Also included are solid form preparations which are intended to beconverted, shortly before use, to liquid form preparations for oraladministration. Such liquid forms include solutions, suspensions, andemulsions. These preparations may contain, in addition to the activecomponent, colorants, flavors, stabilizers, buffers, artificial andnatural sweeteners, dispersants, thickeners, solubilizing agents, andthe like.

A variety of solid, semisolid and liquid vehicles have been known in theart for years for topical application of agents to the skin. Suchvehicles include creams, lotions, gels, balms, oils, ointments andsprays. See, e.g., Provost C. “Transparent oil-water gels: a review,”Int J Cosmet Sci. 8:233-247 (1986), Katz and Poulsen, Concepts inbiochemical pharmacology, part I. In: Brodie B B, Gilette J R, eds.Handbook of Experimental Pharmacology. Vol. 28. New York, N.Y.:Springer; 107-174 (1971), and Hadgcraft, “Recent progress in theformulation of vehicles for topical applications,” Br J Dermatol.,81:386-389 (1972). A number of topical formulations of analgesics,including capsaicin (e.g., Capsin®), so-called “counter-irritants”(e.g., Icy-Hot®, substances such as menthol, oil of wintergreen,camphor, or eucalyptus oil compounds which, when applied to skin over anarea presumably alter or off-set pain in joints or muscles served by thesame nerves) and salicylates (e.g. BenGay®), are known and can bereadily adapted for topical administration of sEHI by replacing theactive ingredient or ingredient with an sEHI, with or without EETs. Itis presumed that the person of skill is familiar with these variousvehicles and preparations and they need not be described in detailherein.

The agent that increases epoxygenated fatty acids (e.g., an inhibitor ofsEH, an EET, an epoxygenated fatty acid, and mixtures thereof),optionally mixed with an anti-inflammatory and/or analgesic agent, canbe mixed into such modalities (creams, lotions, gels, etc.) for topicaladministration. In general, the concentration of the agents provides agradient which drives the agent into the skin. Standard ways ofdetermining flux of drugs into the skin, as well as for modifying agentsto speed or slow their delivery into the skin are well known in the artand taught, for example, in Osborne and Amann, eds., Topical DrugDelivery Formulations, Marcel Dekker, 1989. The use of dermal drugdelivery agents in particular is taught in, for example, Ghosh et al.,eds., Transdermal and Topical Drug Delivery Systems, CRC Press, (BocaRaton, Fla., 1997).

In some embodiments, the agents are in a cream. Typically, the creamcomprises one or more hydrophobic lipids, with other agents to improvethe “feel” of the cream or to provide other useful characteristics. Inone embodiment, for example, a cream may contain 0.01 mg to 10 mg ofsEHI, with or without one or more EETs, per gram of cream in a white tooff-white, opaque cream base of purified water USP, white petrolatumUSP, stearyl alcohol NF, propylene glycol USP, polysorbate 60 NF, cetylalcohol NF, and benzoic acid USP 0.2% as a preservative. In variousembodiments, sEHI can be mixed into a commercially available cream,Vanicream® (Pharmaceutical Specialties, Inc., Rochester, Minn.)comprising purified water, white petrolatum, cetearyl alcohol andceteareth-20, sorbitol solution, propylene glycol, simethicone, glycerylmonostearate, polyethylene glycol monostearate, sorbic acid and BHT.

In other embodiments, the agent or agents are in a lotion. Typicallotions comprise, for example, water, mineral oil, petrolatum, sorbitolsolution, stearic acid, lanolin, lanolin alcohol, cetyl alcohol,glyceryl stearate/PEG-100 stearate, triethanolamine, dimethicone,propylene glycol, microcrystalline wax, tri (PPG-3 myristyl ether)citrate, disodium EDTA, methylparaben, ethylparaben, propylparaben,xanthan gum, butylparaben, and methyldibromo glutaronitrile.

In some embodiments, the agent is, or agents are, in an oil, such asjojoba oil. In some embodiments, the agent is, or agents are, in anointment, which may, for example, white petrolatum, hydrophilicpetrolatum, anhydrous lanolin, hydrous lanolin, or polyethylene glycol.In some embodiments, the agent is, or agents are, in a spray, whichtypically comprise an alcohol and a propellant. If absorption throughthe skin needs to be enhanced, the spray may optionally contain, forexample, isopropyl myristate.

Whatever the form in which the agents that inhibit sEH are topicallyadministered (that is, whether by solid, liquid, lotion, gel, spray,etc.), in various embodiments they are administered at a dosage of about0.01 mg to 10 mg per 10 cm². An exemplary dose for systemicadministration of an inhibitor of sEH is from about 0.001 μg/kg to about100 mg/kg body weight of the mammal. In various embodiments, dose andfrequency of administration of an sEH inhibitor are selected to produceplasma concentrations within the range of 2.5 μM and 30 nM.

The agent that increases epoxygenated fatty acids (e.g., an inhibitor ofsEH, an EET, an epoxygenated fatty acid, and mixtures thereof),optionally mixed with an anti-inflammatory and/or analgesic agent, canbe introduced into the bowel by use of a suppository. As is known in theart, suppositories are solid compositions of various sizes and shapesintended for introduction into body cavities. Typically, the suppositorycomprises a medication, which is released into the immediate area fromthe suppository. Typically, suppositories are made using a fatty base,such as cocoa butter, that melts at body temperature, or a water-solubleor miscible base, such as glycerinated gelatin or polyethylene glycol.

The pharmaceutical preparation is preferably in unit dosage form. Insuch form the preparation is subdivided into unit doses containingappropriate quantities of the active component. The unit dosage form canbe a packaged preparation, the package containing discrete quantities ofpreparation, such as packeted tablets, capsules, and powders in vials orampoules. Also, the unit dosage form can be a capsule, tablet, cachet,or lozenge itself, or it can be the appropriate number of any of thesein packaged form.

The term “unit dosage form”, as used in the specification, refers tophysically discrete units suitable as unitary dosages for human subjectsand animals, each unit containing a predetermined quantity of activematerial calculated to produce the desired pharmaceutical effect inassociation with the required pharmaceutical diluent, carrier orvehicle. The specifications for the novel unit dosage forms of thisinvention are dictated by and directly dependent on (a) the uniquecharacteristics of the active material and the particular effect to beachieved and (b) the limitations inherent in the art of compounding suchan active material for use in humans and animals, as disclosed in detailin this specification.

A therapeutically effective amount or a sub-therapeutic amount of theagent that increases epoxygenated fatty acids can be co-administeredwith a population of stem cells. The dosage of the specific compoundsdepends on many factors that are well known to those skilled in the art.They include for example, the route of administration and the potency ofthe particular compound. An exemplary dose is from about 0.001 μg/kg toabout 100 mg/kg body weight of the mammal. Determination of an effectiveamount is well within the capability of those skilled in the art,especially in light of the detailed disclosure provided herein.Generally, an efficacious or effective amount of a combination of one ormore polypeptides of the present invention is determined by firstadministering a low dose or small amount of a polypeptide or compositionand then incrementally increasing the administered dose or dosages,adding a second or third medication as needed, until a desired effect ofis observed in the treated subject with minimal or no toxic sideeffects. Applicable methods for determining an appropriate dose anddosing schedule for administration of a combination of the presentinvention are described, for example, in Goodman and Gilman's ThePharmacological Basis of Therapeutics, 12th Edition, 2010, McGraw-HillProfessional; in a Physicians' Desk Reference (PDR), 68^(th) Edition,2014, PDR Network; in Remington: The Science and Practice of Pharmacy,21^(st) Ed., 2005, supra; and in Martindale: The Complete DrugReference, Sweetman, 2005, London: Pharmaceutical Press., and inMartindale, Martindale: The Extra Pharmacopoeia, 31st Edition., 1996,Amer Pharmaceutical Assn, each of which are hereby incorporated hereinby reference.

EETs, EpDPEs, or EpETEs are unstable, and can be converted to thecorresponding diols, in acidic conditions, such as those in the stomach.To avoid this, EETs, EpDPEs, or EpETEs can be administered intravenouslyor by injection. EETs, EpDPEs, or EpETEs intended for oraladministration can be encapsulated in a coating that protects thecompounds during passage through the stomach. For example, the EETs,EpDPEs, or EpETEs can be provided with a so-called “enteric” coating,such as those used for some brands of aspirin, or embedded in aformulation. Such enteric coatings and formulations are well known inthe art. In some formulations, the compositions are embedded in aslow-release formulation to facilitate administration of the agents overtime.

It is understood that, like all drugs, sEHIs have half-lives defined bythe rate at which they are metabolized by or excreted from the body, andthat the sEHIs will have a period following administration during whichthey are present in amounts sufficient to be effective. If EETs, EpDPEs,or EpETEs are administered after the sEHI is administered, therefore, itis desirable that the EETs, EpDPEs, or EpETEs be administered during theperiod during which the sEHI are present in amounts to be effective indelaying hydrolysis of the EETs, EpDPEs, or EpETEs. Typically, the EETs,EpDPEs, or EpETEs are administered within 48 hours of administering ansEH inhibitor. Preferably, the EETs, EpDPEs, or EpETEs are administeredwithin 24 hours of the sEHI, and even more preferably within 12 hours.In increasing order of desirability, the EETs, EpDPEs, or EpETEs areadministered within 10, 8, 6, 4, 2, hours, 1 hour, or one half hourafter administration of the inhibitor. When co-administered, the EETs,EpDPEs, or EpETEs are preferably administered concurrently with thesEHI.

6. Methods of Monitoring

Clinical efficacy can be monitored using any method known in the art.Measurable parameters to monitor efficacy will depend on the conditionbeing treated. For monitoring the status or improvement of one or moresymptoms associated with cardiomyopathy, measurable parameters caninclude without limitation, auditory inspection (e.g., using astethoscope), blood pressure, electrocardiogram (EKG), magneticresonance imaging (MRI), changes in blood markers, and behavioralchanges in the subject (e.g., appetite, the ability to eat solid foods,grooming, sociability, energy levels, increased activity levels, weightgain, exhibition of increased comfort). These parameters can be measuredusing any methods known in the art. In varying embodiments, thedifferent parameters can be assigned a score. Further, the scores of twoor more parameters can be combined to provide an index for the subject.

Observation of the stabilization, improvement and/or reversal of one ormore symptoms or parameters by a measurable amount indicates that thetreatment or prevention regime is efficacious. Observation of theprogression, increase or exacerbation of one or more symptoms indicatesthat the treatment or prevention regime is not efficacious. For example,in the case of cardiomyopathy, observation of the improvement of cardiacfunction (e.g., blood pressure in appropriate range, stable heart rhythmor reduction or absence of arrhythmias, changes in blood markers, and/orbehavioral changes in the subject (e.g., increased appetite, the abilityto eat solid foods, improved/increased grooming, improved/increasedsociability, increased energy levels, improved/increased activitylevels, weight gain and/or stabilization, exhibition of increasedcomfort) after one or more co-administrations of stem cells with anagent indicates that the treatment or prevention regime is efficacious.Likewise, observation of reduction or decline of cardiac function (e.g.,blood pressure in appropriate range, unstable heart rhythm or continuedpresence or increased arrhythmias, changes in blood markers, and/orbehavioral changes in the subject (e.g., decreased appetite, theinability to eat solid foods, decreased grooming, decreased sociability,decreased energy levels, decreased activity levels, weight loss,exhibition of increased discomfort) after one or more co-administrationsof stem cells with an agent indicates that the treatment or preventionregime is not efficacious.

In certain embodiments, the monitoring methods can entail determining abaseline value of a measurable biomarker or disease parameter in asubject before administering a dosage of the one or more active agentsdescribed herein, and comparing this with a value for the samemeasurable biomarker or parameter after a course of treatment.

In other methods, a control value (i.e., a mean and standard deviation)of the measurable biomarker or parameter is determined for a controlpopulation. In certain embodiments, the individuals in the controlpopulation have not received prior treatment and do not have the diseasecondition subject to treatment (e.g., cardiomyopathy), nor are at riskof developing the disease condition subject to treatment (e.g.,cardiomyopathy). In such cases, if the value of the measurable biomarkeror clinical parameter approaches the control value, then treatment isconsidered efficacious. In other embodiments, the individuals in thecontrol population have not received prior treatment and have beendiagnosed with the disease condition subject to treatment (e.g.,cardiomyopathy). In such cases, if the value of the measurable biomarkeror clinical parameter approaches the control value, then treatment isconsidered inefficacious.

In other methods, a subject who is not presently receiving treatment buthas undergone a previous course of treatment is monitored for one ormore of the biomarkers or clinical parameters to determine whether aresumption of treatment is required. The measured value of one or moreof the biomarkers or clinical parameters in the subject can be comparedwith a value previously achieved in the subject after a previous courseof treatment. Alternatively, the value measured in the subject can becompared with a control value (mean plus standard deviation) determinedin population of subjects after undergoing a course of treatment.Alternatively, the measured value in the subject can be compared with acontrol value in populations of prophylactically treated subjects whoremain free of symptoms of disease, or populations of therapeuticallytreated subjects who show amelioration of disease characteristics. Insuch cases, if the value of the measurable biomarker or clinicalparameter approaches the control value, then treatment is consideredefficacious and need not be resumed. In all of these cases, asignificant difference relative to the control level (i.e., more than astandard deviation) is an indicator that treatment should be resumed inthe subject.

7. Kits

Further provided are kits, stents and patches comprising a population ofstem cells and one or more agents that increase the production or levelsof EETs. Embodiments of the stem cells and one or more agents thatincrease the production or levels of EETs are as described above andherein. In some embodiments, the stem cells are provided in anappropriate container, e.g., a vial, a tube, a pouch, a bag. Stem cellcoated or loaded stents are known in the art and find use, e.g., in themethods and kits, e.g., as described in Raina, et al., Heart. 2014 June;100(Suppl 3):A88-A89; Savchenko, et al., Vestn Rentgenol Radiol. 2013July-August; (4):41-6; Wang, et al., Cardiovasc Res. 2009 Dec. 1;84(3):461-9; Motwani, et al., Biotechnol Appl Biochem. 2011January-February; 58(1):2-13; Wu, et al., J Biomed Mater Res A. 2011Sep. 1; 98(3):442-9; and Intl. Patent Publ. No. WO 2008/094936. Patches,including cardiac patches, which can serve as repositories for thedelivery of stem cells are also known in the art and find use, e.g., inthe methods and kits, e.g., as described in Kim, et al., Integr Biol(Camb). 2012 September; 4(9):1019-33; LeBlanc, et al., Stem Cells Trans'Med. 2013 November; 2(11):896-905; Lam, et al., Tissue Eng Part A. 2013March; 19(5-6):738-47; Wang, et al., Antioxid Redox Signal. 2014 Apr.30; Wickham, et al, J Biomed Mater Res B Appl Biomater. 2014 Mar. 24;and Martinez-Ramos, et al., Tissue Eng Part C Methods. 2014 Mar. 14.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 Beneficial Effects of sEHIs on Cardiac Remodeling Post MI

We have demonstrated the beneficial effects of several sEHIs in cardiacremodeling in clinically relevant models of cardiac hypertrophy,fibrosis and failure (1-8). A sEHI, TPPU (1-trifluoromethoxyphenyl-3-(1-propionylpiperidine-4-yl) urea, FIG. 2 a) was started 1 weekpost MI (FIG. 2 b). Treatment with TPPU resulted in a significantreduction in cardiac dilatation and the amount of collagen depositioncompared to MI alone (FIG. 2 c, d and e). A single-cell based assayusing flow cytometry was used to label myocytes (MC) withcardiac-specific troponin T antibody (FIG. 3A). Cardiac fibroblasts wereidentified as Thy1.2+ and lin−/CD31/CD45/CD34− cells (FIGS. 3A and B)(23). The findings demonstrated a significant increase in Thy1.2+ cellsin the remote area from the infarct zone in MI mice compared to shamanimals. Treatment with TPPU resulted in a significant decrease inThy1.2+ fibroblasts (FIG. 3C) and a significant decrease inproliferative fibroblasts (Ki67+) (FIG. 3D) compared to MI alone.

Example 2 Mechanisms Underlying the Observed Beneficial Effects of sEHIsin the Heart

Our studies provide new insights into the mechanisms of action of sEHIsin cardiac hypertrophy and failure: 1) Using metabolomic profiling, wedemonstrate a significant decrease in the EETs/DHETs ratio afterthoracic aortic banding and MI, suggesting a heightened inflammatorystate; 2) Treatment with sEHIs results in the normalization of theEETs/DHETs ratio and a concomitant reduction in inflammatory cytokinesand chemokines (FIGS. 4 a & b)(5); 3) A significant reduction in myocytehypertrophy and apoptosis (5) and finally; 4) Inhibition of theactivation of NF-kB pathway in cardiac myocytes (1)

Research Design and Methods:

This example tests the survival, engraftment, integration, and functionof transplanted hiPSC-CMs in the acute model of MI with and withoutsEHIs treatment.

Tissue injury from MI results in robust inflammatory responses,involving the synthesis and release of chemo- and cytokines and therecruitment of fibroblasts, which may form physical barriers at the siteof injury and inhibit stem cell integration and survival. Our recentresults have demonstrated that sEHIs reduce pro-inflammatory cytokinesand chemokines (IL-6, IFN-γ, TNF-α and MCP-1, FIG. 4) and areanti-fibrotic, capable of reducing collagen deposition in the injuredmyocardium (8). Therefore, treatment with sEHIs in the MI model resultsin the improvement in the survival and functional integration ofhiPSC-CMs in NSG mice. To test, inhibitors of sEH enzyme wereadministered in a murine myocardial infarction (MI) model. In addition,the role of sEH enzyme was directly tested using gene-targeted sEH nullmutant mice (24).

Experimental Design:

HiPSCs containing the puromycin gene under the α-myosin heavy chainpromoter were transduced with a double fusion construct of reportergenes; firefly luciferase for bioluminescence imaging and greenfluorescent protein (GFP) (25). GFP+hiPSCs were sorted using florescentactivated cell sorter (FACS) (FIG. 5), differentiated into beatingclusters of hiPSC-CMs (26) and enriched with puromycin treatment.

Mouse MI Model:

MI were generated in 10- to 12-week-old male NSG and sEH null mutant(sEH^(−/−)) mice as we have previously described (8). One week after thesurgery, NSG mice were randomized into six different groups: 1)Sham±sEHI, 2) MI±sEHI and 3) MI+hiPSC-CM±sEHI. Transplantation ofhiPSC-CMs into the border zones (27) was performed using ultrasound(VisualSonics Vevo 2100) guided injection and mice were treated withsEHIs (15 mg/L) in drinking water one week after surgery for a period of3 weeks. Similarly, a total of 6 groups were required to test theeffects of genetic deletion of sEH (Ephx2) including: 1) Sham, 2) MI, 3)MI+hiPSC-CM in Ephx2^(−/−) animals compared to wild-type (WT)littermates. Cardiac fibrosis was evaluated using hydroxyproline assay,histology, flow cytometry, and immunofluorescence confocal imaging as wehave previously described (8). All the animals will undergo in vivoexperimentation for 3 weeks at the end of which the animals weresacrificed for in vitro analyses.

I. In Vivo Analyses:

Longitudinal In Vivo Bioluminescence Imaging (BLI).

To quantify the survival and engraftment of transplanted hiPSC-CMs overtime, in vivo BLI imaging was performed at the UC Davis Center forMolecular and Genomic Imaging using the IVIS Xenogen system. Ourexciting BLI data shows a significant improvement in the survival ofGFP+hiPSC-CMs in NSG mice with MI at week 4 with sEHI treatment vs. nosEHI treatment (FIG. 6A-B). Quantification by flow cytometry showeddouble the number of GFP+hiPSC-CMs in the sEHI treated mice (10±0.2%)compared to the untreated mice (5±1%) (FIG. 6C).

Functional analysis by echocardiography was performed to evaluate thesystolic and diastolic function. We have obtained exciting data from sixgroups of animals (FIG. 7) demonstrating a significant improvement inthe fractional shortening (FS) as assessed using motion-modeechocardiography in the MI+hiPSC-CM+sEHI group (62±2.6%) compared toMI+sEHI (55±0.6%), MI+hiPSC-CMs (55±1.1%), and MI alone (45±0.5%) groupsat 4 week (FIG. 7, *P<0.05, ANOVA). As expected, there was a decrease inthe FS in the MI group from one week (51±1.1%) to 4 week (45±0.5%),suggesting adverse remodeling in the MI alone group. Indeed, treatmentwith sEHI and hiPSC-CMs in the MI animal resulted in an addedimprovement in the fractional shortening (FS) compared with the MIalone.

In vivo hemodynamic monitoring was performed using a four-electrode PVcatheter (Millar Instruments) to record chamber volume by impedance andpressure by micromanometry (28). Different parameters were assessedincluding LV end diastolic pressure. PV loops were constructed beforeand during transient reduction of preload to generate specific systolicand diastolic function indexes, LV afterload (indexed by arterialelastance), ejection fraction, contractile function as assessed throughload-independent parameters (maximal power index & preload recruitablestroke work), & diastolic function (LV stiffness constant, tau and peakrate of pressure decline (dP/dt_(min)) (FIG. 8) (28, 29).

In vivo electrophysiologic studies (5, 30) were performed to test forinducible cardiac arrhythmias and finally optical imaging was performedto assess conduction velocity and action potential duration (FIG. 9)(31-33). The spectrally distinct GFP was excited to imagehiPSC-CMs-derived cells from the epicardial surface to assess theengraftment and integration.

II. In Vitro Analyses:

Histology:

To quantify cardiac fibrosis, cardiac sections from six groups ofanimals were examined using Masson's Trichrome stain. Our data with thesix groups of animals shows a decrease in fibrosis in the hiPSC-CM+sEHIgroup compared to the MI alone mice (FIG. 10A).

Immunofluorescence Confocal Microscopy:

The engraftment and integration of transplanted hiPSC-CMs were assessedusing immunofluorescence confocal microscopy and histology to detectdonor cells (GFP+) and cardiac-specific cell types as well as theintegration between transplanted hiPSC-CMs and host cardiomyocytes usingantibodies specific for connexin. Our data shows an increased presenceof GFP+ cells in the MI+sEHI mice compared to MI alone (FIG. 10B).

Single-cell based assays using flow cytometry were performed asdescribed previously (34) to quantify the number and proliferativecapacity of GFP+hiPSC-CMs using troponin T (cTnT) antibodies anddifferent populations of cardiac fibroblasts using various markers(Thy1.2, FSP-1, DDR2) (23, 35-39). The hiPSC-CM volume were analyzedusing Coulter Multisizer 4 as previously described (40).

Metabolic profiling of oxylipin levels were performed using LC-MS/MS aswe have described (1-7). The increase in the EETs/DHETs ratios willdirectly document the target engagement by sEHIs.

Second harmonic generation (SHG) microscopy, a novel imagining techniquewere used to image and quantify the myofilament, structures, anddynamics to assess the contractility and maturation of hiPSC-CMnon-invasively (FIG. 11). SHG microscopy is a non-linear label-freetechnique that can directly image the stem cell derived sarcomeres basedon the unique intrinsic property of the myosin rod domains to generate asignal at twice the frequency and half the wavelength of the laser beamused to excite the sarcomeres.

Example 3 Directly Testing the Molecular Mechanisms Underlying theBeneficial Effects of sEHIs on Stem Cell Survival and Integration

Oxidative stress activates Erk1/2 and NF-κB, which further causesapoptosis and reducing the oxidative stress shows an increased stem cellengraftment (Song, et al., Stem Cells. 2010; 28:555-563; Tusi, et al.,Biomaterials. 2011; 32:5438-5458; Ahmad, et al., Toxicology Letters.2012; 208:149-161). The nuclear translocation of NF-κB is required forapoptosis as well (Tusi, et al., Biomaterials. 2011; 32:5438-5458;Ahmad, et al., Toxicology Letters. 2012; 208:149-161). EETs have beenshown to reduce oxidative stress and apoptosis in other organs (Chen, etal., Cell Physiol Biochem. 2014; 33(6):1663-80; Li, et al., MolPharmacol. 2013 December; 84(6):925-34). Here, we determined whethertreatment with sEHIs reduces oxidative stress and apoptosis in the hostmyocardial cells as well as the engrafted hiPSC-CMs by inactivatingErk1/2 and NF-κB. To test, we directly examine the oxidative stress(production of ROS) and apoptosis in the cardiomyocytes (CMs),non-myocyte cells (NMCs) and transplanted hiPSC-CMs in the sEHI treatedand non-treated groups.

In addition, the mechanistic basis for the observed beneficial effectswas tested in the transplanted hiPSC-CMs. EETs regulate gene expressionby maintaining NF-κB in an inactive state (1, 19). Since sEH enzymeconverts EETs to inactive DHET, increased levels of EETs by treatmentwith sEHIs prevent the activation and nuclear localization of NF-κB inthe transplanted hiPSC-CMs.

Experimental Design:

Two sEHIs with significantly different chemical structures (TPPUcontaining the piperidine ring and t-AUCB containing the adamantanering) were used (Table 2, supra).

Apoptosis Assay:

The degree of apoptosis was analyzed using single-cell based flowcytometry in the NMCs, cardiomyocytes (CM) and transplantedGFP+hiPSC-CMs in sEHI treated and non-treated mice as describedpreviously (34) using propidium iodide and Annexin V (LifeTechnologies). Our apoptosis assay data in all cardiac cells indicatesthat there was a significant decrease in apoptosis in NMCs and CMsfractions in the MI+hiPSC-CMs+sEHI group compared to the MI,MI+hiPSC-CMs, MI+sEHI groups (FIG. 12). Our exciting analysis in thetransplanted GFP+hiPSC-CMs shows a significant decrease in apoptoticcells in the MI+hiPSC-CMs+sEHI group compared to MI+hiPSC-CMs withoutsEHI treatment (FIG. 13).

Oxidative Stress Assay:

The oxidative stress in the myocardium was analyzed using isolatedsingle cells for the production of ROS using flow cytometry. Thecell-permeant CellRox (Life technologies) probe remains non-fluorescentwhile in a reduced state and exhibits bright fluorescence upon oxidationby ROS. Our assay data in all cardiac cells indicates that there was asignificant decrease in the ROS production in NMCs and CM fractions inthe MI+hiPSC-CMs+sEHI group compared to the MI, MI+hiPSC-CMs, MI+sEHIgroups (FIG. 14). In addition, we have obtained additional data in thetransplanted GFP+hiPSC-CMs which demonstrate a significant decrease inROS production in the MI+hiPSC-CMs+sEHI group compared to MI+hiPSC-CMswithout sEHI treatment (FIG. 15).

Activation of NF-κB:

NF-κB represents one of the critical players in the cytokine-mediatedinflammation. NF-κB was maintained in the inactive form when bound toIκB, which was degraded by IκB kinase. Degradation of IκB releases theNF-κB dimer from the cytosol leading to nuclear translocation of NF-κBand gene activation. Since sEHIs prevent the conversion of EETs and EETsin turn inhibit IκB kinase (19), treatment with sEHI may prevent theactivation and nuclear translocation of NF-κB in the transplanted stemcells exposed to inflammatory cytokines Indeed, we have obtainedexciting data that demonstrate an increased nuclear translocation ofNF-κB in the cultured hiPSC-CMs upon TNF-α stimulation (20 ng/ml for 20min). This effect was inhibited by the treatment of sEHI (FIG. 16). Wewill further test the activation of NF-κB in the in vivo models of MIusing confocal microscopy and western blotting as previously described(1). We also tested whether sEHIs can block the activation of NF-κBusing Western blot analysis (FIG. 16B). Total IκB and phosphorylated-IκB(pIκB) levels assessed after TNF-α stimulation showed a decrease in IκBlevel and an increase in the pIκB levels associated with the activationof NF-κB. TNF-α stimulation showed an increase in the level of NF-κB inthe nuclear fraction, which was not seen in sEHI treated cells (FIGS.16C and 16D).

Statistical analyses: All data were tested for normality by theShapiro-Wilk test and homogeneity of variances were assessed using theLevene's test (42). Repeated measures of analysis of variance (RM-ANOVA)combined with post hoc analyses were performed with treatment as abetween-group variable.

Treatment with sEHIs results in an increase in the survival,engraftment, and integration of transplanted hiPSC-CMs with an increasein the formation of gap junctions between transplanted hiPSC-CMs andhost cardiac myocytes compared to the treatment with hiPSC-CMs alone.There was a concomitant decrease in fibrosis associated with animprovement in cardiac function and a decrease in inducible arrhythmias.At the in vivo level, this translates into an improvement in cardiacfunction associated with a reduction in arrhythmia inducibility. Theremay be immune rejection of the transplanted stem cells in the sEH nullmutant mice. This can be decreased with the use of immunosuppression inthe hiPSC-CMs treated sEH null mutant mice. Mechanistically, treatmentwith sEHIs results in a decrease in cellular apoptosis, as well as adecrease in ROS production and NF-κB activation in transplantedhiPSC-CMs.

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It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof are suggested to persons skilled in the art and are tobe included within the spirit and purview of this application and scopeof the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1. A method of increasing, improving and/or promoting the survival,engraftment, and/or integration of transplanted stem cells in a tissueof a subject in need thereof, comprising co-administering to the subjectthe stem cells with an agent that increases the production and/or levelof epoxygenated fatty acids.
 2. A method of reversing, mitigating and/orimproving one or more symptoms associated with cardiomyopathy or cardiacarrhythmia in an subject in need thereof, said method comprisingco-administering to said subject a population of stem cells and an agentthat increases the production and/or level of epoxygenated fatty acids.3. The method of claim 1, wherein the subject has cardiomyopathy, andwherein the cardiomyopathy is one or more of hypertrophiccardiomyopathy, hypertensive cardiomyopathy, diabetic cardiomyopathy anddilated cardiomyopathy.
 4. (canceled)
 5. The method of claim 2, whereinsaid cardiomyopathy is due to or secondary to one or more of valvularheart disease, myocardial infarction, and familial hypertrophiccardiomyopathy, and wherein said valvular heart disease is secondary torheumatic fever, myxomatous degeneration of the valve, or papillarymuscle dysfunction.
 6. (canceled)
 7. The method of claim 2, wherein thecardiomyopathy is dilated cardiomyopathy, and wherein said dilatedcardiomyopathy is one or more of alcohol-induced cardiomyopathy,viral-induced cardiomyopathy, familial dilated cardiomyopathy anddilated cardiomyopathy is caused by administration of an anti-cancerdrug or exposure to a toxic agent.
 8. (canceled)
 9. The method of claim2, wherein the administration of said stem cells and said agent oragents inhibits cardiac arrhythmia, wherein the cardiac arrhythmia isone or more of atrial fibrillation, atrial flutter, ventricularfibrillation and ventricular tachycardia.
 10. (canceled)
 11. The methodof claim 1, wherein the agent comprises one or more epoxygenated fattyacids.
 12. The method of claim 1, wherein the epoxygenated fatty acidsare selected from the group consisting of cis-epoxyeicosantrienoic acids(“EETs”), epoxides of linoleic acid, epoxides of eicosapentaenoic acid(“EPA”), epoxides of docosahexaenoic acid (“DHA”), epoxides of thearachidonic acid (“AA”), epoxides of cis-7,10,13,16,19-docosapentaenoicacid, and mixtures thereof.
 13. (canceled)
 14. A method of claim 1,wherein the agent is an inhibitor of soluble epoxide hydrolase (“sEH”).15. The method of claim 14, wherein the inhibitor of sEH comprises aprimary pharmacophore selected from the group consisting of a urea, acarbamate, and an amide. 16-19. (canceled)
 20. The method of claim 14,wherein the inhibitor of sEH has an 1050 of less than about 100 μM.21-26. (canceled)
 27. The method of claim 1, wherein the subject is ahuman.
 28. The method of claim 1, wherein the stem cells are selectedfrom multipotent stem cells, pluripotent stem cells, and inducedpluripotent stem cells.
 29. The method of claim 1, wherein the stemcells comprise mesenchymal stem cells, myocyte stem cells and/orcardiomyocyte stem cells. 30-31. (canceled)
 32. The method of claim 1,wherein the stem cells are selected from the group consisting ofcardiomyocytes or cardiac progenitor cells derived from multipotent stemcells, cardiomyocytes or cardiac progenitor cells derived frompluripotent stem cells, cardiomyocytes or cardiac progenitor cellsderived from induced pluripotent stem cells, adult cardiac progenitorcells and cardiac stem cells, and wherein the stem cells comprise adultcardiac stem cells or cardiac progenitor cells derived from humancardiac tissues.
 33. (canceled)
 34. The method of claim 1, wherein thestem cells are syngeneic, allogeneic or xenogeneic to the subject.35-37. (canceled)
 38. The method of claim 1, wherein the stem cells areadministered intravenously, intra-arterially or intralesionally. 39.(canceled)
 40. The method of claim 1, wherein the stem cells and theagent that increases the production and/or level of epoxygenated fattyacids are administered by different routes of administration.
 41. Themethod of claim 14, wherein the stem cells and the inhibitor of sEH areconcurrently co-administered.
 42. The method of claim 14, wherein thestem cells and the inhibitor of sEH are sequentially co-administered.43. The method of claim 1, wherein the tissue is cardiac tissue. 44.(canceled)
 45. A stent comprising a population of stem cells and one ormore agents that increase the production and/or level of epoxygenatedfatty acids.
 46. (canceled)
 47. A patch comprising a population of stemcells and one or more agents that increase the production and/or levelof epoxygenated fatty acids. 48-69. (canceled)